purification, characterization, and kinetic analysis of a 55-kda form

THE JOURNAL 0 1992 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 267, No. 23, Issue of August 15, PP. 16297-16304,1992 Printed in U.S.A.

Purification, Characterization, and Kinetic Analysis of a 55-kDa Form of Phosphatidylinositol 4-Kinase from Saccharomyces cereuisiae*

(Received for publication, April 6, 1992)

Joseph T. Nickels, Jr., Rosa J. Buxeda, and George M. Carman$ From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903

A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4- phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q- Sepharose, Mono Q, and hydroxylapatite 11. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 RIM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 OC. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X- lOO/phosphatidylinositol- mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The in- terfacial Michaelis constant (K,) and the dissociation constant (KB) for phosphatidylinositol in the Triton X- 100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The K,,, for MgATP was 0.36 mM. 55- kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.

* This work was supported by United States Public Health Service Grant GM-35655 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. This is New Jersey Agricultural Experiment Station Publication D- 10541-1-92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ T O whom correspondence and reprint requests should be ad- dressed.

The synthesis and turnover of the phosphoinositides PIP’ and PIP, play a significant role in intracellular signaling in higher eucaryotic organisms (1, 2). Because of its tractable molecular genetic system, the yeast Saccharomyces cereuisiae is being used as a model eucaryote to examine the regulation of phosphoinositide metabolism. PIP and PIP2 were first discovered in S. cereuisiae by Lester and Steiner (3). These phosphoinositides are localized to the plasma membrane (4), and their metabolism is regulated by glucose (5,6), ATP levels (4, 5), and sterol (7, 8). Studies using antibodies directed against PIP2 have shown that PIPz or its hydrolysis products play an essential role in S. cereuisiae proliferation (9).

PI 4-kinase (ATP:phosphatidylinositol4-phosphotransferase, EC 2.7.1.67) catalyzes the formation of PIP and ADP from PI and ATP (10). This enzyme catalyzes the committed step in the synthesis of the phosphoinositides in S. cereuisiae (11). Thus, the regulation of PI 4-kinase activity should play a role in phosphoinositide turnover and cell growth. PI 4- kinase activity is activated when wild-type (12) and sterol auxotrophic (13) cells enter the GI phase of the cell cycle. Studies using mutants defective in the RASIcAMP pathway suggested that PI 4-kinase activity is also activated by CAMP- dependent protein kinase (14).

Our laboratory has taken the approach of purifying PI 4- kinase so that studies on the mechanism and regulation of enzyme activity could be conducted in a well defined system. We previously purified a 45-kDa form of PI 4-kinase from microsomal membranes and characterized its enzymological and kinetic properties (11,15). In this paper we report on the purification to near homogeneity of a second form of PI 4- kinase which had a molecular mass of 55-kDa. The physicochemical and enzymological properties of the 55-kDa PI 4- kinase differed from those of the 45-kDa PI 4-kinase. Fur- thermore, results of defined kinetic experiments showed that the two forms of PI 4-kinase differed with respect to their affinities to PI and in their catalytic binding to PI. These studies raised the suggestion that the two forms of PI 4-kinase may be regulated differentially in uiuo.

EXPERIMENTAL PROCEDURES

Materials

All chemicals were reagent grade. Nucleotides, phospholipids, PMSF, TPCK-trypsin, protease Staphylococcus aureus V8, molecular mass standards for glycerol gradient centrifugation, and bovine serum albumin were purchased from Sigma. Radiochemicals were purchased fron Du Pont-New England Nuclear. Scintillation counting supplies were purchased from National Diagnostics. Triton X-100 was a gift

The abbreviations used are: PIP, phosphatidylinositol 4-phosphate; PIP,, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; TPCK, ~-l-tosylamido-2-phenylethyl chloromethyl ke- tone; PMSF, phenylmethylsulfonyl fluoride; CDTA, trans-1,2-dia- minocyclohexane-N,N,N’,N’-tetraacetic acid; SDS, sodium dodecyl sulfate.

16297

16298 Yeast 55-kDa PI 4-Kinase from Rohm and Haas Co. DE52 (DEAE-cellulose) and silica gel- loaded SG8l chromatography paper were purchased from Whatman, Inc. Silica Gel 60 thin-layer chromatography plates were from EM Science. Hydroxylapatite (Bio-Gel HT), molecular mass standards for electrophoresis, electrophoresis reagents, isoelectric focusing reagents, and immunochemical reagents were purchased from Bio-Rad. Q-Sepharose and the Mono Q chromatography column were from Pharmacia LKB Biotechnology, Inc. 3ZP-Labeled PIP was prepared as described previously (11).

Methods

Growth Conditions

Wild-type S. cerevisiae strain ade5 MATa (16, 17) was used for enzyme purification. The organism was maintained on YEPD (1% yeast extract, 2% peptone, 2% dextrose) media plates containing 2% Bacto-agar. Cells were grown in YEPD medium a t 28 "C to late exponential phase, harvested by centrifugation, and stored at -80 "C (18).

Purification of the 45-kDa PI 4-Kinase

The 45-kDa PI 4-kinase was purified to near homogeneity as described previously (11, 15). The specific activity of the purified enzyme was 3.9 pmol/min/mg.

Enzyme Assays and Protein Determination

PI 4-kinase activity was measured for 10 min at 30 "C by following the phosphorylation of PI with [y-32P]ATP (10,000-15,000 cpm/ nmol) as described previously (19). The reaction mixture for the 55- kDa PI 4-kinase contained 50 mM Tris maleate buffer (pH 7.0), 2.5 mM ATP, 10 mM MgC12, 0.2 mM PI, and 3.2 mM Triton x-100 in a total volume of 0.1 ml. The reaction mixture for the 45-kDa PI 4- kinase contained 50 mM Tris-HC1 buffer (pH 8.0), 2.5 mM ATP, 30 mM MgC12, 0.2 mM PI, and 12.5 mM Triton X-100 in a total volume of 0.1 ml. All assays were conducted in triplicate with an average standard deviation of & 5 %. All assays were linear with time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 pmol of product/min. Specific activity was defined as units per mg of protein. Protein concentration was determined by the method of Bradford (20) using bovine serum albumin as the standard.

Product Identification

The 32P-labeled phospholipid products of the reactions catalyzed by the 55- and 45-kDa PI 4-kinases were extracted with chloroform (19) and analyzed by chromatography on CDTA-treated silica gel plates (21) using solvent system A and EDTA-treated SG81 paper (22) using solvent system B. Solvent system A (21) contained chloroform, methanol, pyridine, formic acid, water (2025:15:1:2.5) and 4 g of boric acid, and solvent system B (22) contained chloroform, methanol, and 2.5 M ammonium hydroxide (9:7:2). The positions of the labeled products on the chromatograms were determined by autoradiography and compared with standard phospholipids.

Electrophoresis, Isoelectric Focusing, and Immunoblotting Native polyacrylamide gel electrophoresis (23) was performed at

5 "C using 6% slab gels containing 0.5% Triton X-100. SDS-polyacrylamide gel electrophoresis (24) was performed with 10% slab gels. Proteins on polyacrylamide gels were visualized by the silver stain procedure (25) or with Coomassie Blue. Isoelectric focusing (26) was performed as described by Morlock et al. (27). Immunoblot analyses (28, 29) of cell extracts and purified enzyme preparations were performed with purified antibodies to the 45-kDa PI 4-kinase protein (15).

Proteolysis

SDS-polyacrylamide gel slices containing the 55- and 45-kDa forms of PI 4-kinase were subjected to proteolysis using 100 ng of S. aureus V8 and 100 ng of TPCK-trypsin as described by Cleveland et al. (30). The proteolysis products were separated by SDS-polyacrylamide gel electrophoresis using 15% gels. The gels were doubled stained with Coomassie Blue and silver.

Amino Acid Analysis

The 55- and 45-kDa forms of PI 4-kinase on SDS-polyacrylamide gels were transferred to polyvinylidene difluoride paper and subjected

to amino acid composition analysis (31) by Mark P. Flocco at the Princeton University Microchemistry Core Facility.

Preparation and Analysis of Triton X-IOOIPI-mixed Micelles

PI in chloroform was transferred to a test tube, and solvent was removed in uacuo for 40 min. Uniform Triton X-lOO/PI-mixed micelles were prepared by adding Triton X-100 to the dried phospholipid and were analyzed by glycerol gradient centrifugation (15).

Glycerol Gradient Centrifugation

Glycerol gradient centrifugation was performed as described previously (15) using 5 2 0 % glycerol gradients. The buffer system used throughout the gradient was 50 mM Tris-HC1 (pH 8.0), 0.35 mM Triton X-100, and 10 mM MgC1,.

Purification of 55-kDa PI 4-Kinase

All steps were performed at 5-8 "C. Step I: Preparation of Cell Extract-The cell extract was prepared

from 200 g (wet weight) of cells by disruption with glass beads in 50 mM Tris maleate (pH 7.0), 1 mM Na,EDTA, 0.3 M sucrose, 10 mM 2- mercaptoethanol, and 1 mM PMSF (11).

Step 2: Preparation of Microsomes-Microsome membranes were isolated from the cell extract by differential centrifugation (18). Microsomes were washed in Buffer A (50 mM Tris-HC1 (pH 8.0), 10 mM MgC12, 10 mM 2-mercaptoethanol, 10% glycerol, and 1 mM PMSF) and frozen at -80 "C until used for purification.

Step 3: Preparation of Triton X-I00 Extract-PI 4-kinase activity was extracted from microsomes using 1% Triton X-100 in Buffer A at a final protein concentration of 10 mg/ml(11). PI 4-kinase activity was very labile after solubilization with Triton X-100 and was used immediately for the next step in the purification.

Step 4: DE52 Chromatography-A DE52 column (2.5 X 5 cm) was equilibrated with Buffer B (50 mM Tris-HC1 (pH 8.0), 10 mM MgCl,, 10 mM 2-mercaptoethanol, 10% glycerol, 1 mM PMSF, and 0.5% Triton X-100). The Triton X-100 extract was applied to the column followed by the washing of the column with 5 column volumes of Buffer B. PI 4-kinase activity was eluted from the DE52 column with Buffer B containing 0.3 M NaCl a t a flow rate of 50 ml/h. A step elution was used to expedite the recovery of the enzyme due to activity lability at this stage in the purification. Fractions containing PI 4- kinase activity were pooled and immediately used for the next step in the purification scheme.

Step 5: Hydroxylapatite Chromatography I-A hydroxylapatite column (2.5 X 3 cm) was equilibrated with Buffer C (10 mM potassium phosphate (pH 8.0), 10 mM MgCI,, 10 mM 2-mercaptoethanol, 10% glycerol, 1 PMSF, and 0.5% Triton X-100). The enzyme preparation from the previous step was diluted 4:l with Buffer C and applied to the column. The column was washed with 3 column volumes of Buffer C followed by elution of the enzyme with 10 column volumes of a linear potassium phosphate gradient (0.01-0.3 M) in Buffer C at a flow rate of 15 ml/h. Two peaks of PI 4-kinase activity eluted from the column at phosphate concentrations of about 0.04 and 0.12 M. The most active fractions under the second peak of PI 4-kinase activity were pooled and dialyzed against Buffer B. The hydroxylapatite enzyme preparation was completely stable and could be frozen at -80 "C until used for the next step in the purification scheme.

Step 6: Q-Sepharose Chromatography-A Q-Sepharose column (2.5 x 7 cm) was equilibrated with Buffer B. Enzyme from the previous step was applied to the column at a flow rate of 25 ml/h. The column was washed with 10 column volumes of Buffer B followed by elution of the enzyme with 10 column volumes of a linear NaCl gradient (0- 0.3 M) in Buffer B. The peak of PI 4-kinase activity eluted from the column at 0.17 M NaCl. The most active fractions were pooled and dialyzed against Buffer B.

Step 7: Mono Q Chromatography-A Mono Q column (0.5 X 5 cm) was equilibrated with Buffer B. Q-Sepharose-purified enzyme was applied to the column at a flow rate of 60 ml/h. The column was washed with 10 column volumes of Buffer B followed by elution of the enzyme with 40 column volumes of a linear NaCl gradient (0- 0.25 M) in Buffer B. The peak of PI 4-kinase activity eluted at a NaCl concentration of 0.18 M. Fractions containing activity were pooled and dialyzed against Buffer C.

Step 8: Hydroxylapatite Chromatography 11-A second hydroxylapatite column (1.5 X 7 cm) was equilibrated with Buffer C. Enzyme from the Mono Q step was applied to the column at a flow rate of 5 ml/h. The column was washed with 10 volumes of Buffer C. PI 4- kinase activity was eluted from the column with 10 volumes of a linear potassium phosphate gradient (0-0.2 M) in Buffer C. The peak

Yeast 55-kDa PI 4-Kinase 16299

of PI 4-kinase activity eluted at 0.18 M potassium phosphate. Frac- tions containing activity were pooled and stored at -80 "C. The purified enzyme was completely stable for at least 2 months.

RESULTS

Purification of 55-kDa PI 4-Kinase

A summary of the purification of the 55-kDa PI 4-kinase is shown in Table I. PI 4-kinase activity was solubilized from the microsomes with 1% Triton X-100 followed by purification in the presence of Triton X-100 by conventional chromatography steps. The hydroxylapatite I (10.3-fold) and the Mono Q (36.7-fold) chromatography steps afforded the great- est enrichments in specific activity in the purification. In addition to providing a 10-fold purification of the enzyme, the hydroxylapatite I chromatography step separated the 55-kDa PI 4-kinase from the 45-kDa form of the enzyme. Two peaks of PI 4-kinase activity eluted from the hydroxylapatite I column (Fig. 1). The enzyme under the second peak of activity was the 55-kDa PI 4-kinase, whereas the enzyme under the first peak of activity was the 45-kDa form of the enzyme (11, 15). Overall, the enzyme was purified 10,166-fold over the cell extract with an activity yield of 6.5% to a final specific activity of 5 pmol/min/mg (Table I).

The purification scheme resulted in the isolation of a major peptide band with an apparent minimum subunit molecular mass of 55 kDa as shown by SDS-polyacrylamide gel electrophoresis (Fig. 2). We were unable to measure PI 4-kinase activity from gel slices after SDS-polyacrylamide gel electrophoresis or on nitrocellulose blots following electrophoretic transfer of the protein from SDS-polyacrylamide gels (32). To confirm that the 55-kDa protein was indeed PI 4-kinase, we used a combination of native and SDS-polyacrylamide gel electrophoresis and measuring activity from gel slices (11). The purified enzyme was subjected to native polyacrylamide gel electrophoresis in the presence of 0.5% Triton X-100 at 5 "C. The presence of Triton X-100 in the native gel was required to recover enzymatic activity. Following electrophoresis, one lane from a slab gel was stained with silver and showed a protein with an RF of 0.4. The protein from the native gel was excised, denatured, and subjected to SDS- polyacrylamide gel electrophoresis. This analysis showed one peptide band of 55 kDa similar to that shown in Fig. 2. Gel slices from a duplicate lane of the native slab gel containing the purified enzyme were minced with a razor blade and homogenized in assay buffer at 5 "C. PI 4-kinase activity was measured at 30 "C using [T-~'P]ATP, and the chloroform- soluble product was extracted. The chloroform-soluble counts from the reaction were at least 20,000 cpm above background counts derived from gel slices not containing protein. The chloroform-soluble product of the reaction comigrated with standard PIP using solvent systems A and B (Fig. 3). Solvent system A separates PIP from PI 3-phosphate based on the

ability of PIP but not PI 3-phosphate to form a complex with boric acid (21). The product of the 45-kDa PI 4-kinase, previously identified as PIP (11, 15), was included in the analysis as a control.

Physicochemical Properties of 55-kDa PI 4-Kinase

To examine differences between the 55- and 45-kDa forms of PI 4-kinase, amino acid composition, isoelectric focusing, and peptide mapping analyses were performed. The amino acid compositions of the 55- and 45-kDa forms of PI 4-kinase are presented in Table 11. The amino acid compositions of the two enzymes were different, although some amino acids were present a t similar frequences. The isoelectric points of the 55- and 45-kDa enzymes were 5.02 and 6.53, respectively. The proteolysis patterns of the 55- and 45-kDa proteins with TPCK-trypsin and S. aureus V8 are shown in Fig. 4. Treat- ment of the two forms of PI 4-kinase with TPCK-trypsin and V8 yielded different peptide patterns. The most obvious differences are indicated by arrows in the figure.

Affinity-purified antibodies to the 45-kDa PI 4-kinase (15) were used to examine if the 55- and 45-kDa forms of PI 4- kinase were immunologically related. Purified samples of the 55- and 45-kDa enzymes were subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose paper and subjected to immunoblot analysis using anti-45-kDa PI 4-kinase antibodies. The anti-45-kDa PI 4-kinase antibodies only detected the 45-kDa enzyme and did not cross-react with the 55-kDa enzyme. These antibodies do not detect the 55-kDa PI 4- kinase in cell extracts (15).

Enzymological Properties of 55-kDa PI 4-Kinase

Dependence of 55-kDa PI 4-Kinase on pH, Magnesium, Manganese, and Triton X-100"Maximum 55-kDa PI 4-kinase activity was dependent on 10 mM magnesium ions (Fig. 5B) and 3.2 mM Triton X-100 (molar ratio of Triton X-100 to PI of 16:l) (Fig. 50) at the pH optimum of 7.0 (Fig. 5A). The magnesium ion dependence could be substituted by 0.5 mM manganese ions (Fig. 5C). The apparent inhibition of 55- kDa PI 4-kinase activity at concentrations of Triton X-100 above 3.2 mM is characteristic of surface dilution kinetics (33). The role of Triton X-100 in the assay for 55-kDa PI 4- kinase is to form a mixed micelle with PI providing a surface for catalysis (see below).

Effect of Temperature on 55-kDa PI 4-Kinase Actiuity-55- kDa PI 4-kinase activity was measured from 0 to 50 "C (Fig. 6A). Maximum activity was observed at 30 "C. An Arrhenius plot for 55-kDa PI 4-kinase was constructed (Fig. 6B) and used to calculate an activation energy for the reaction of 12.1 kcal/mol. The enzyme was examined for its stability to temperatures ranging from 30 to 70 "C (Fig. 6C). 55-kDa PI 4- kinase activity was unstable above 30 "C with total inactiva-

TABLE I Purification of 55-kDa PI 4-kinase

55-kDa PI 4-kinase was purified from S. cereuisiae as described under "Experimental Procedures." The data are based on starting with 200 g (wet weight) of cells.

Purification step Total units Protein Specific activity Yield Purification

rrnol/min 1. Cell extract 4.670 2. Microsomes 3.571 3. Triton X-100 extract 4. DE52

3.204 2.753

5. Hydroxylapatite I 1.977 6. Q-Sepharose 2.025 7. Mono Q 1.133 8. Hydroxylapatite I1 0.305

mg 9,340 2,880 1,343

972 67.8 36.7

0.56 0.06

unitslmg 0.0005 0.0012 0.0024 0.0028 0.0292 0.0552 2.0178 5.0833

7% -fold 100 1 76.4 2.4 68.6 4.8 58.9 5.6 42.3 58.4 43.3 110.6 24.2 4,035.7 6.5 10,166.6

16300 Yeast 55-kDa PI 4-Kinase

n 40 z _” 30 E

i

5 10

‘=. 20

0

Y

a - 0

’ 1 0.4 I I

0 J 0.0 0 10 20 30 40

Fraction Number

FIG. 1. Elution profile of PI 4-kinase activity after hydroxylapatite chromatography I. Fractions (I ml) were collected and assayed for 55-kDa PI 4-kinase activity (0) and protein (--) as described under “Experimental Procedures.” The phosphate gradient profile is indicated by the dashed line. The PI 4-kinase activity under the first peak was 45-kDa PI 4-kinase. The 45-kDa PI 4-kinase activity was an underestimate since activity was measured under the assay conditions for the 55-kDa PI 4-kinase.

1 2 -v-

.<*e

“- - 55 kDa

* 1

lmmmmi -PI

I ’ “PIP2

B l 2

; “PIP2

FIG. 3. Identification of the phospholipid product catalyzed by 55-kDa PI 4-kinase. Native-polyacrylamide gel slices containing purified 55-kDa PI 4-kinase were assayed for PI 4-kinase activity using [y-32P]ATP as described under “Experimental Procedures.” The 3zP-labeled phospholipid product of the reaction was extracted with chloroform and analyzed by thin-layer chromatography using solvent systems A (panel A ) and B (panel B ) . The product of the reaction catalyzed by 55-kDa PI 4-kinase is shown in lane 1. The product of the 45-kDa PI 4-kinase reaction was used as a control and is shown in lane 2. The positions of phosphoinositide standards are indicated.

TABLE I1 Amino acid comvositions of the 55- and 45-kDa forms of PI 4 -k ime

Amino acid PI 4-kinase

55 kDa 45 kDa

I “

ir

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified 55-kDa PI 4-kinase. Purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis as described under “Experimental Procedures.” Lane 1 shows a Coomassie Blue- stained polyacrylamide gel of protein molecular mass standards: phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa). Lane 2 shows a Coomassie Blue-stained polyacrylamide gel of purified 55-kDa PI 4-kinase.

tion of the enzyme after heating for 10 min at 60 “C. The tl/2 value for the inactivation of 55-kDa PI 4-kinase at 40 “C was calculated to be 2.1 min (Fig. 6D).

Inhibitors of 55-kDa PI 4-Kinase Activity-55-kDa PI 4- kinase was inhibited by various thioreactive compounds (Table 111). The addition of 2-mercaptoethanol to the assay system blocked this inhibition. The enzyme was also inhibited by MgADP (Fig. 7) and calcium ions (Fig. 8). IC5o values for MgADP and calcium were calculated to be 0.5 and 5.6 mM, respectively.

Kinetic Properties of 55-kDa PI 4-Kinase

Dependence of 55-kDa PI 4-Kinase on PI in Triton X-lOO/ PI-mixed Micelles-Detergentlphospholipid-mixed micelle systems permit defined kinetic analyses in an environment

Asx Glx Ser

Thr Ala ‘4% TY Val Met Phe Ile Leu Pro Lvs

G ~ Y

residues f monomer

42.9 51 10.1 44.1 45.5 29.1 54.1 52.1 2.5 2.4

48.4 42.6 34.1 10.3 6.6 15.3

29.6 5.4 1.2 5.6

16.6 24.1 23.5 26.8 41.2 20.5 9.1 5.2 1 .a ND”

ND, not detected.

that mimics the physiological surface of the membrane (34, 35). A kinetic analysis of the 55-kDa PI 4-kinase was performed with Triton X-lOO/PI-mixed micelles according to the “surface dilution” (33) and “dual phospholipid” (36, 37) kinetic models. These kinetic models have been useful for the kinetic analysis of several phospholipid-dependent enzymes (37-42) including the 45-kDa PI 4-kinase (15). Both kinetic models are similar in that the enzyme is postulated to asso- ciate with the Triton X-100 micelle surface followed by the formation of an enzyme-substrate complex, catalysis, and release’of products. The difference between the two kinetic models is the first step involving the binding of the enzyme to the micelle surface. In the surface dilution (33) kinetic model the enzyme nonspecifically binds to the micelle surface, whereas in the dual phospholipid (37) kinetic model the

Yeast 55-kDa PI 4-Kinase 1 2 3 4 5 6

16301

FIG. 4. Proteolysis of the 55-kDa and 45-kDa forms of PI 4-kinase with TPCK-trypsin and S. aureus V8. SDS-polyacrylamide gel slices containing about 1 pg of the 55-kDa (lanes I and 4) and about 1 pg of the 45-kDa (lanes 3 and 6 ) forms of PI 4-kinase were digested with TPCK-trypsin (lanes 1 and 3 ) and S. aureus V8 (lanes 4 and 6 ) . Peptides were separated on 15% SDS-polyacrylamide gels and visualized by double staining with Coomassie Blue and silver. TPCK-trypsin (lane 2 ) and V8 (lane 5) were run alone. The positions of the protein molecular mass standards bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa) are indicated. Peptides observed from one form of PI 4-kinase that were not observed from the other form of PI 4-kinase are indicated by arrows.

I I I I I ' B I e 2 Q!

0

Y ._ h 2l 1 e 2Y-o 1 I

0- 0- 4 6 8 1 0 0 10 20

pH Magnesium, mM

I I I

0 1 2 Manganese. mM

0 15 30 Triton X-100, mM

FIG. 5. Effect of pH, magnesium, manganese, and Triton X- 100 on 55-kDa PI 4-kinase activity. PI 4-kinase activity was measured at the indicated pH values with 50 mM Tris maleate buffer (pH 5.0-7.5), Tris-HC1 buffer (pH 7.0-9.0), or glycine-NaOH buffer (pH 9.0-10.0) (panel A) ; the indicated concentrations of MgC12 (panel B ) ; the indicated concentrations of MnC12 (panel C); and the indicated concentrations of Triton X-100 (panel D).

enzyme binds specifically to its substrate in the micelle surface.

To obtain physical data for the association of 55-kDa PI 4- kinase with Triton X-100 micelles and Triton X-lOO/PI- mixed micelles, we examined the sedimentation profiles of the enzyme after glycerol gradient centrifugation. Pure Triton X-100 micelles sediment near the top of the gradient, whereas Triton X-lOO/PI-mixed micelles sediment with a size slightly larger than pure Triton X-100 micelles (15). 55-kDa PI 4- kinase associated with Triton X-100 micelles in the absence (Fig. 9A) and the presence of PI (Fig. 9B) . Thus the surface dilution and dual phospholipid kinetic models should be ap- plicable to the 55-kDa PI 4-kinase.

The applicability of these kinetic models for the 55-kDa PI 4-kinase was examined in detail as described for the 45-kDa PI 4-kinase (15). 55-kDa PI 4-kinase activity was measured

0 20 40 Temperature.'C

20 40 60 Temperature."C

-" 2 0.1 .- L

01

0 .- Y -0.3 a

-I -0.7

- OI

3.2 3.4 3.6

1 / O K x lo-'

-I 0 4 8

lime, min

FIG. 6. Effect of temperature on 55-kDa PI 4-kinase activity. Panel A, PI 4-kinase activity was measured at the indicated temperatures under standard assay conditions in a controlled temperature water bath. Panel B, the data in panel A from 0 to 30 "C were plotted as log PI 4-kinase activity versus the reciprocal of the absolute temperature (l/K). Panel C, samples of the 55-kDa PI 4- kinase were incubated at the indicated temperatures for 10 min. After incubation the samples were cooled on ice followed by the measurement of activity a t 30 "C. Panel D, samples of the 55-kDa PI 4-kinase were incubated at 40 "C for the indicated time intervals. After incubation the samples were cooled on ice followed by the measurement of activity a t 30 "C. The curves drawn in panels B and D were the result of least squares analysis of the data.

TABLE I11 Effect of thioreactive compounds on 55-kDa PI 4-kinase activity PI 4-kinase activity was measured under standard assay conditions

in the Dresence of the indicated additions.

Component Relative activity

5%

Component Relative activity

5% Control 100 +5 mM HgCI2 1 +5 mM HgC1, + 10 mM 2-mercaptoethanol 60 +5 mM p-chloromercuriphenylsulfonic acid 1 +5 mM p-chloromercuriphenylsulfonic acid + 10 102

+5 mM N-ethylmaleimide 1 +5 mM N-ethylmaleimide + 10 mM 2-mercapto- 83

mM 2-mercaptoethanol

ethanol

100 c -I

0 0.0 0.4 0.8 1.2

MgADP, rnM

FIG. 7. Effect of MgADP on 55-kDa PI 4-kinase activity. PI 4-kinase activity was measured under standard assay conditions with 0.3 mM MgATP in the presence of the indicated concentrations of MgADP.

as a function of the sum of the molar concentrations of Triton X-100 and PI (Fig. 10, surface dilution model) and as a function of the molar concentration of PI (Fig. 11, dual phospholipid model) at a series of set surface (mol fraction)


1

\ L11 25 1

0- 0 10 20 30

Colcium, mM

FIG. 8. Effect of calcium on 55-kDa PI 4-kinase activity. PI 4-kinase activity was measured under standard assay conditions in the presence of the indicated concentrations of CaC12.

Lo 0.4

0 7

x 0.2 E - \

=- 0.0 ai

.E 0.6

VJ 0

Y - a 0.3

0.0

0.8

$ 0.4 2

2 0.0 g

7

I 0.8

0 Y

0.4

0.0 0 5 10 15 Bottom TOP

Fraction Number

FIG. 9. Association of 55-kDa PI 4-kinase with Triton X- 100 micelles and Triton X-lOO/PI-mixed micelles after glycerol gradient centrifugation. Samples (0.3 ml) of 55-kDa PI 4- kinase and 3 mM Triton X-100 ( A ) and 55-kDa PI 4-kinase, 3 mM Triton X-100, and 0.2 mM PI (mol fraction of PI of 0.06) ( B ) were subjected to glycerol gradient centrifugation as described under “Ex- perimental Procedures.” The amount of 55-kDa PI 4-kinase activity that was applied to the gradients was 2 X unit. PI 4-kinase activity (0) was measured as described under “Experimental Proce- dures,” and Triton X-100 (0) was monitored by measuring its ab- sorbance at 275 nm. The yields of 55-kDa PI 4-kinase activity from the glycerol gradients shown in panels A and B were 21 and 35%, respectively. The positions of the molecular mass standards (apofer- ritin (443 kDa), 0-amylase (200 kDa), and bovine serum albumin (66 kDa)) from bottom to top are indicated in panel A .

concentrations of PI. Activity was dependent on the bulk concentrations of Triton X-100 plus PI (Fig. 10) and the bulk concentration of PI (Fig. 11) at each surface concentration of PI. In each case, as the surface concentration of PI in the mixed micelle decreased, there was a decrease in the apparent VmaX (Figs. 10 and 11). Furthermore, the double-reciprocal plots of the data in Figs. 10 and 11 showed that 55-kDa PI 4- kinase displayed saturation kinetics when the bulk concentration of substrate was varied at each fixed surface concentration of PI. The pattern of lines shown in Figs. 10 and 11 were indicative of an equilibrium-ordered step as predicted by the surface dilution (33) and dual phospholipid (37) kinetic models. Replots (15,33) of the l/Vintercepts uersus 1/PI mol fraction from Figs. 10 and 11 were linear and used to calculate the kinetic constants V,,, and K,,,. The V,,, and K , values for 55-kDa PI 4-kinase for each model were the same, 2.2 pmol/min/mg and 0.013 mol fraction (1.3 mol %), respectively. Replots (15, 33) of the slopes uersus 1/PI mol fraction from the data in Figs. 10 and 11 were also linear and used to

4 PI/TX + PI

rn 0.004 V 0.0038

A 0.015

0 0.03 > ‘ 1 2

0

-1.5 0.0 1.5

1/[TX + PI], mM”

FIG. 10. Activity of the 55-kDa PI 4-kinase toward PI in mixed micelles with Triton X-100 according to the surface dilution kinetic model. PI 4-kinase activity was measured as a function of the sum of the molar concentrations of Triton X-100 plus PI at set mol fractions of PI. The data are plotted as 1 /V (units/mg) versus the reciprocal of the sum of the concentration of Triton X-100 (TX) plus PI. The lines drawn are a result of a least squares analysis of the data.

V 0.0038

rn 0.009 > A 0.01 5

0 0.03

1

0

-50 -25 0 25 50

l /PI, mM-’

FIG. 11. Activity of the 55-kDa PI 4-kinase toward PI in mixed micelles with Triton X- 100 according to the dual phospholipid kinetic model. PI 4-kinase activity was measured as a function of the molar concentration of PI at set mol fractions of PI. The data are plotted as 1/V (units/mg) uersus the reciprocal of the PI concentration. The lines drawn are a result of a least squares analysis of the data.

calculate the K, values for the enzyme according to each model. The K, values for the Triton X-lOO/PI-mixed micelle (surface dilution model) and for PI (dual phospholipid model) were 11.8 and 0.035 mM, respectively.

The 55-kDa PI 4-kinase fit the surface dilution and dual phospholipid kinetic models equally well. Both analyses yielded the same V,,, and K , values. The results of these analyses suggested that 55-kDa PI 4-kinase had more affinity for the Triton X-100 micelle when PI was present. This was reflected in the lower K, value (0.035 mM) determined according to the dual phospholipid model when compared with the relatively high K, value (11.8 mM) determined according to the surface dilution model.

Dependence of 55-kDa PI 4-Kinase on ATP and PI and Reaction Mechanism-In our kinetic experiments to examine the dependence of 55-kDa PI 4-kinase on ATP and PI the enzyme was measured such that activity was only dependent on the surface concentration of PI (i.e. at a bulk PI concentration of 0.2 mM, Fig. 11). 55-kDa PI 4-kinase activity exhibited saturation kinetics when the MgATP concentration was varied at various fixed surface concentrations of PI (Fig. 12A) and when the surface concentration of PI was varied at various fixed concentrations of MgATP (Fig. 13A). Replots (43) of these data were linear (Figs. 12B and 13B) and used to calculate K,,, values for PI and MgATP of 0.017 mol fraction (1.7 mol %) and 0.36 mM, respectively. The K,,, value determined for PI by this kinetic analysis agreed well with the K,,, values determined for PI according to the surface dilution and dual phospholipid kinetic models.

Yeast 55-kDa PI 4-Kinase 16303

The intersecting lines in the reciprocal plots shown in Figs. 12A and 13A were consistent with the 55-kDa PI 4-kinase catalyzing a sequential Bi Bi reaction (43). In sequential Bi Bi reactions the two substrates must bind to the enzyme before catalysis and release of either product (43). Thus for the 55-kDa PI 4-kinase reaction PI and ATP must bind to the enzyme before the release of PIP and ADP. To determine the order of substrate binding and product release in the reaction, we examined the ability of the 55-kDa PI 4-kinase to catalyze isotopic exchange reactions (43). According to Cleland (43), the labeled product that shows exchange into one substrate in the absence of the other product is the first product released in the reaction sequence. 55-kDa PI 4-kinase catalyzed the exchange between the 3'P-labeled phosphate moiety of inositol of PIP and the y-phosphate of ATP in the presence of PI (Table IV, reaction 1). The exchange of the labeled phosphate from ["PIPIP into water-soluble material was dependent on ATP since the enzyme did not catalyze the hydrolysis of ["PIPIP (Table IV, reaction 2). The enzyme also catalyzed the exchange of the 3ZP-labeled y-phosphate of ATP into PIP in the presence of ADP (Table IV, reaction 3). The exchange of the labeled y-phosphate from [3zP]ATP into chloroform-soluble material was not caused by the formation of PIPz (Table IV, reaction 4). The results of the isotopic exchange reactions suggested that PIP was released prior to ADP in the forward reaction, and ATP was released before P I in the reverse reaction.

2 PI/TX + PI

0.004 A 0.006 e 0.00s v 0.01 0 0.05

< F

1

0

-3 0 3 6

1 /MgATP, mM" 0.4

u a m 0 0.2 -

0.0 -100 0 100 200

[PI/TX + PI]"

FIG. 12. Dependence of the 55-kDa PI 4-kinase activity on MgATP using Triton X-lOO/PI-mixed micelles. PI 4-kinase activity was measured as a function of the molar Concentration of MgATP at set mol fractions of PI. The molar concentration of PI was 0.2 mM. Panel A , the data are plotted as I / V (units/mg) versus the reciprocal of the MgATP concentration. Panel B, replot of the 1/ V intercepts obtained in panel A versus the reciprocal of the mol fraction of PI. The lines drawn are a result of a least squares analysis of the data.

-150 0 150 300 450

[PI/TX + PI]-'

MgATP, mM + 0.1 A 0.2

0.4 v 0.8

0.02

Iu

- m 0" 0.01

0.00

- 3 0 3 6 9

1 /MgATP. mM"

FIG. 13. Dependence of the 55-kDa PI 4-kinase activity on the surface concentration of PI using Triton X-lOO/PI-mixed micelles. PI 4-kinase activity was measured as a function of the mol fraction of PI at set molar concentrations of MgATP. The molar concentration of PI was held constant at 0.2 mM while the Triton X- 100 concentration was varied. Panel A , the data are plotted as 1 /V (units/mg) versus the reciprocal of the mol fraction of PI. Panel B, replot of the slopes obtained in panel A versus the reciprocal of the MgATP concentration. The lines drawn are a result of a least squares analysis of the data.

DISCUSSION

Preliminary studies in our laboratory suggested that two forms of membrane-associated PI 4-kinase existed in S. cere- vis& (44). This was based on the observation that PI 4- kinase had two pH optima in microsomal membranes. We subsequently purified a 45-kDa form of PI 4-kinase (11, 15). We now report on the purification of a 55-kDa form of the enzyme. The 45-kDa PI 4-kinase could be separated from the 55-kDa form of the enzyme by DE52 chromatography. 45- kDa PI 4-kinase elutes from the DE52 column with 0.1 M NaCl (11, 45), whereas the 55-kDa PI 4-kinase eluted from the column with 0.3 M NaCI. Because of the lability of both forms of PI 4-kinase during DE52 chromatography, we step- eluted both enzymes from the column with 0.3 M NaC1. The two forms of the enzyme were then separated on the first hydroxylapatite column. The eight-step purification scheme developed here for the 55-kDa PI 4-kinase resulted in a nearly homogeneous enzyme preparation. The final specific activity and fold purification of the 55-kDa PI 4-kinase were similar to those of the 45-kDa PI 4-kinase (11, 15).

TABLE IV Reactions catalyzed by 55-kDa PI 4-kinase

Reactions were measured in the standard assay buffer described under "Experimental Procedures." Reactions were run for 24 h with 1 X IO" unit of pure enzyme and the indicated reaction components. ATP-PIP exchange reactions were measured with either (y3'P]ATP (10,000 cpm/nmol) or ["PIPIP (200 cpm/nmol) as substrate. Water- soluble radioactive ATP and Pi and chloroform-soluble radioactive PIP and PIP, were measured after a chloroform/methanol/water phase partition (11).

Reaction Incorporated Apparent or released exchange

nmol % 1. 0.2 mM PI + 2.5 mM ATP + 0.2 mM 7.56 37

2. 0.2 mM [32P]PIP hydrolysis NR" 0 3. 2.5 mM ADP + 0.2 mM PIP + 2.5 2.37 12

["PIPIP

mM [y3'P]ATP 4. 0.2 mM PIP + 2.5 mM f-p3'P1ATP NR 0

a NR, no reaction.


TABLE V Enzymological and kinetic properties of the 55- and 45-kDa forms of

PI 4-kinase PI 4-kinase

55 kDa 45 kDa" Property

pH optimum 7.0 8.0 Cofactor dependence

Mg2+ 10 mM Mn2+

27 mM 0.5 mM

Triton X-1OO:PI depend- 161 62.51

Activation energy 12.1 kcal/mol 31.5 kcal/mol K. (micelle) Ks (PI) Km (PI) Km (ATP)

ence

11.8 mM 118 mM 0.035 mM 0.26 mM 1.7 mol % 0.36 mol % 0.36 mM 0.5 mM

Data are taken from Refs. 11 and 15.

Multiple forms of PI 4-kinase have been purified and characterized from several animal sources with molecular masses ranging from 45 to 76 kDa (46-52). The basic enzymological properties of the yeast 55-kDa PI 4-kinase were generally similar to those of the 55-kDa PI 4-kinase from higher eu- caryotes (47,48,50-52). The reports on the PI 4-kinases from animal sources do not address the physicochemical properties nor the detailed kinetics that were reported here and previously (11, 15) for the yeast enzymes.

In addition to having different subunit molecular masses, the 55-and 45-kDa forms of PI 4-kinase from S. cereuisiue differed with respect to their amino acid compositions, isoelectric points, and proteolytic peptide fragments. Further- more, the two enzymes were not immunologically related as determined using affinity-purified antibodies directed against the 45-kDa P I 4-kinase. The 55- and 45-kDa forms of PI 4- kinase also differed in several enzymological and kinetic properties (Table V). The 55-kDa PI 4-kinase differed from the 45-kDa PI 4-kinase with respect to pH optimum, dependence on divalent cations, Triton X-100, and activation energy. Both forms of the enzyme differed in their association to Triton X- 100 micelles and Triton X-lOO/PI-mixed micelles as well as their catalytic binding to PI in Triton X-100 micelles. The 55-kDa PI 4-kinase had a 7-lo-fold greater affinity (reflected in the K, values) for the Triton X-100 micelle surface when compared with the affinity for the micelle of the 45-kDa PI 4-kinase (15). However, once bound to PI in the micelle surface, the 45-kDa PI 4-kinase had a 4.7-fold greater catalytic efficiency (reflected in the K , values) for PI (15) than did the 55-kDa PI 4-kinase.

The differences in the enzymological and kinetic properties of the two forms of PI 4-kinase raised the suggestion that these enzymes may be regulated differentially in uiuo. Fur- thermore, these differences could be exploited to measure differentially the relative activities of each enzyme in membrane preparations. The availability of the pure 55- and 45- kDa forms of PI 4-kinase will facilitate studies on the regulation of PI 4-kinase activity in S. cereuisiue. For example, we can now address which form of the PI 4-kinase is regulated by phosphorylation (14) and by the growth phase of cells (12, 13). Furthermore, since mutants defective in PI 4-kinase do not exist, the pure enzymes will facilitate the cloning and molecular characterization of the genes encoding for these enzymes.

Acknowledgment-We thank Mark Flocco for performing the amino acid compositional analyses of the enzymes.

REFERENCES

2. Downes, C. P., and Macphee, C. H. (1990) Eur. J . Biochem. 193 , 1-18 1. Berridge, M. J. (1987) Biochim. Biophys. Acta 907 , 33-45

3. Lester, R. L., and Steiner, M. R. (1968) J. Biol. Chem. 243 , 4889-4893 4. Patton, J. L., and Lester, R. L. (1992) Arch. Biochem. Biophys. 2 9 2 , 70-

5. Talwalker, R. T., and Lester, R. L. (1974) Biochim. Biophys. Acta 360, 76

m - x ~ 6. Kaibuchi, K., Miyajima, A,, Arai, K.-I., and Matsumoto, K. (1986) Proc.

7. Dahl, J. S., and Dahl, C. E. (1985) Btochem. Biophys. Res. Commun. 133 ,

." "_

Natl. Acad. Sci. U. S. A. 83,8172-8176

I l A A - I l W Y I 1 -""

8. Dahl, C., Biemann, H.-P., and Dahl, J. (1987) Proc. Natl. Acad. Sci. U. S. A.

9. Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) 84, 4012-4016

Nature 333, 188-190 10. Colodzin, M., and Kennedy, E. P. (1965) J. Biol. Chem. 240 , 3771-3780 11. Belunis, C. J., Bae-Lee, M., Kelley, M. J., and Carman, G. M. (1988) J.

12. Holland, K. M., Homann, M. J., Belunis, C. J., and Carman, G. M. (1988)

13. Duhois, E. L., and Wiame, J.-M. (1978) Mol. & Gen. Genet. 164, 275-283 14. Kato, H., Uno, I., Ishikawa, T., and Takenawa, T. (1989) J. Biol. Chem.

15. Buxeda, R. J., Nickels, J. T., Jr., Belunis, C. J., and Carman, G. M. (1991)

16. Greenherg, M. L., Klig, L. S., Letts, V. A., Loewy, B. S., and Henry, S. A.

17. Klig, L. S., Homann, M. J., Carman, G. M., and Henry, S. A. (1985) J .

18. Fischl, A. S., and Carman, G. M. (1983) J. Bacteriol. 154,304-311

20. Bradford, M. M. (1976) Anal. Biochem. 72 , 248-254 19. McKenzie, M. A,, and Carman, G. M. (1982) J. Food Biochem. 6 , 77-86

21. Walsh, J. P., Caldwell, K. K., and Majerus, P. W. (1991) Proc. Natl. Acad.

22. Steiner, S., and Lester, R. L. (1972) J. Bacteriol. 109, 81-88 23. Davis, B. (1964) Ann. N. Y. Acud. Sei. 121,404-427 24. Laemmli, U. K. (1970) Nature 227,680-685 25. Merril, C. R., Dunau, M. L., and Goldman, D. (1981) Anal. Biochem. 110 ,

26. Ames, G. F.-L., and Nakaido, K. (1976) Biochemistry 15,616-623 27. Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991)

28. Haid, A,, and Suissa, M. (1983) Methods Enzymol. 9 6 , 192-205 29. Fischl, A. S., Homann, M. J., Poole, M. A,, and Carman, G. M. (1986) J.

30. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. Biol. Chem. 261,3178-3183

31. LeGendre, N., and Matsudaira, P. (1989) in A Practical Guide to Protein (1977) J. Biol. Chem. 252 , 1102-1106

and Peptide Purification for Microsequencing (Matsudaira, P. T., ed) pp. 49-69, Academic Press, San Diego

32. Poole, M. A,, Fischl, A. S., and Carman, G. M. (1985) J. Bacteriol. 161 , 772-774

33. Deems, R. A,, Eaton, B. R., and Dennis, E. A. (1975) J. Bid. Chem. 250 ,

Biol. Chem. 263 , 18897-18903

J. Bacteriol. 170 , 828-833

264,3116-3121

J. Bid. Chem. 266,13859-13865

(1983) J. Bacterid. 153 , 791-799

Bacteriol. 162, 1135-1141

Sci. U. S. A. 88,9184-9187

201-207

J. Biol. Chem. 266,3586-3593

m ~ ~ n 9 n 34. Dennis, E. A. (1983) in The Enzymes (Boyer, P. D., ed) Vol. 16, pp. 307-

35. Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260 ,

36. Roberts, M. F., Deems, R. A,, and Dennis, E. A. (1977) Proc. Natl. Acad.

37. Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259 , 5734-

I"" 1""

353, Academic Press, New York

10039-10043

Sci. U. S. A. 74,1950-1954

38. Carman, G. M., and Dowhan, W. (1979) J. Biol. Chem. 254,8391-8397 39. Dowhan, W., Wickner, W. T., and Kennedy, E. P. (1974) J. Biol. Chem.

40. Lin, Y.-P., and Carman, G. M. (1990) J. Bbl. Chem. 265 , 166-170 41. Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5740-

42. Kucera, G. L., Miller, C., Sisson, P. J., Wilcox, R. W., Wiemer, Z., and 5744

43. Cleland, W. W. (1970) in The Enzymes (Boyer, P. D., ed) Vol. 2, pp. 1-65, Waite, M. (1988) J. Biol. Chem. 263,12964-12969

44. McKenzie, M. A. (1982) Partial Purification and Properties of Membrane- Academic Press, New York

associated Phosphatidylinositol Kinase from Saccharomyces cereuisiae. M.S. thesis, Rutgers University, New Brunswick, NJ

5739

249,3079-3084

45. McKenzie, M. A., and Carman, G. M. (1983) J. Bacteriol. 156, 421-423 46. Saltiel, A. R., Fox, J. A,, Sherline, P., Sahyoun, N., and Cuatrecasas, P.

47. Porter, F. D., Li, Y.-S., and Deuel, T. F. (1988) J . Biol. Chem. 263,8989-

48. Walker, D. H., Dougherty, N., and Pike, L. J. (1988) Biochemistry 2 7 ,

49. Yamakawa, A,, and Takenawa, T. (1988) J. Biol. Chem. 2 6 3 , 17555-17560 50. Wetzker, R., Klinger, R., Hsuan, J., Fry, M. J., Kauffmann-Zeh, A., Muller,

E., Frunder, H., and Waterfield, M. (1991) Eur. J . Biochem. 200 , 179-

51. Jenkins, G. H., Suhrahmanyam, G., and Anderson, R. A. (1991) Biochim. 185

52. Scholz, G., Barritt, G. J., and Kwok, F. (1991) Eur. J. Biochem. 201 , 249- Biophys. Acta 1080 , 11-18

255

(1987) Biochem. J . 241 , 759-763

8995

6504-6511

purification, characterization, and kinetic analysis of a 55-kda form

Documents