Biochimica et Biophysica Acta, 358 (1974) 363--366 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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BBA Report

BBA 61296

GLYCOGEN PHOSPHORYLASE ACTIVITY IN PERMEABILIZED CELLS OF S A C C H A R O M Y C E S C E R E V I S A E

JOSE A. ACEVEDO*, AGNES MARTINEZ-LAHOZ and FERMIN SAGARDIA**

Department of Microbiology, University of Puerto R ico, School of Medicine, Rio Piedras, 00936 (U.S.A.)

(Received May 20th, 1974)

Summary

The velocity of formation of glucose 6-phosphate and glucose by protamine-treated permeabilized yeast cells was dependent on the concentra- tion of inorganic phosphate. Fluoride inhibited the reaction. Only small amounts of glucose 6-phosphate were produced in presence of ATP and glucose. We conclude that we are measuring the glycogen phosphorylase reaction inside the permeabilized cells.

We have investigated the possibility of measuring the activity of glycogen phosphoryiase (a-l,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1 [1,2 ] ) in permeabflized cells of Saccharomyces cerevisiae prepared by protamine treatment [3]. These structures, although not viable, retain essentially all of the macromolecular components. Their low molecular weight compounds are lost and their permeability and other surface prop- erties are altered, so that specific enzymic reactions can be measured by incubating them with appropriate substrates and measuring the products in the medium. It is presumed that some of the intracellular relationships are not totally altered, so that information relevant to real intracellular events can be obtained. Such an approach has been attempted with both yeast [3,4] and bacterial [5,6] permeabilized cells prepared by various means.

S. cerevisiae (Guiness Strain 1338) was grown for 48 h in bacto- peptone (5 g/l), yeast extract (4 g/l), malt extract (4 g/l), gluoose (1%) at

* P resen t a d d r e s s : D e p a r t m e n t o f B io logy , Un ive r s i t y o f P u e r t o l~ico, R i o P ied ras , P u e r t o R i c o , U .S .A . ** T o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .

3 6 4

room temperature without aeration. Cells were collected by centrifugation, washed twice with distilled water, lyophilized, and stored in the cold. Permeabilized cells were prepared by incubation with protamine, as prev- iously described [3]. Under phase-contrast microscopy many of the cells exhibited a mosaic-like structure on their outer surface and invaginations of the membrane (Fig. 1). They were not viable, and did not ferment glucose.

Fig.1. G r o u p of p e r m e a b i l i z e d y e a s t cells exhibi t ing a mosaic- l ike a ~ a n g e m e n t on the i r o u t e r surface. A n h ivag inat ion o f t h e m e m b r a n e c a n b e s e e n in o n e o f t h e cells.

Phosphorylase was assayed in the direction of glycogen degradation by shaking a cell suspension (8.0 mg/ml final concentration) in 10 mM Tris, pH 7.5 or 10 mM histidine, pH 6.5 or 6.8 buffers, and 1.0 mM Pi at 30 °C. Aliquots of 5.0 ml were removed at intervals (usually 0, 30, and 60 min), rapidly centrifuged, and the supernatants frozen immediately. Glc-6-P was determined in a coupled assay system by measuring the formation of NADPH at 340 nm in presence of excess glucose-6-phosphate dehydrogenase. The actual system used is a slight modification of that used for the direct assay of phosphorylase [7 ]. Phosphoglucomutase was added to this system for the determination of glucose 1-phosphate. Glucose was determined with the glucostat reagent. Glycogen was extracted from yeast cells with 5 M NaOH followed by 0.5 M HC14 (modified from [8] ), and assayed with the phenol-- sulfuric acid reagent [9]. Part of the glycogen becomes extracted by the base, and must be precipitated with 3 vols of ethanol and assayed separately.

Table I shows that considerable amounts of Glc-6-P and glucose are formed when these cells are incubated with Pi, but that hardly any products are formed in its absence. There was no detectable formation of glucose 1- phosphate, indicating that the equilibrium of the phosphoglucomutase reac- tion inside the cells favors Glc-6-P formation.

Although it did not seem likely that the Glc-6-P could be formed f~om Pi through the formation of ATP, since these cells are largely depleted of intracellular small molecular weight compounds, we investigated this

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T A B L E I

F O R M A T I O N O F G L U C O S E 6 - P H O S P H A T E A N D G L U C O S E IN P E R M E A B I L I Z E D Y E A S T C E L L S

T h e cel l s u s p e n s i o n ( 8 . 0 m g / m l f ina l c o n c e n t r a t i o n ) w a s i n c u b a t e d a t 3 0 ° C in 1 0 m M Trls b u f f e r , 7 .5 . A d d i t i o n s w e r e m a d e t o the fo l lowing final concentrations: Pi, 1 .0 raM; s o d i u m fluoride, 0.1 M; ATP--MgC12 , 0 .1 m M ; g lucose , 0 .1 raM. Velocit ies of format ion of glucose 6-phosphate and glucose are e x p r e s s e d as n a n o m o l e s f o r m e d / m l o f r e a c t i o n m i x t u r e / h .

A d d i t i o n s t o s y s t e m Glc-6-P Glucose

N o n e 0 0 N a F 0 0 PI 1 1 6 1 8 0 P i + N a F 38 0 G l u c o s e + A T P - - M g 2 8 - - O l u c o s e + A T P - - M g + N a F 0

possibility. As Table I shows only small amounts of GIc-6-P were formed in presence of glucose, ATP, and magnesium ions.

The fact that fluoride inhibits the formation of glucose completely indicate that the free sugar is formed by the action of phosphatases. In addi- tion, fluoride causes a partial inhibition of the phosph0rylase reaction, as it also does in vitro [10].

There was no reaction when the supernatants from the ghost sus= pensions were incubated with Pi alone or in combination with glycogen; thus, the enzymatic activity resides inside the cells. Furthermore, there was no reduction of NADP in the assay system unless glucose-6-phosphate dehydro- genase was added, which showed that the results are not due to the formation of unspecific reducing compounds.

When the concentrat ion of Pi was varied from 0.75--4.0 raM, the velocity of product formation (the sum of Glc-6-P and glucose) varied follow- ing hyperbolic kinetics. Km values for Pi at pH values of 6.5--7.5 were in the range of 1.0--2.0 mM. These values are close to those obtained for other phosphorylases in vitro [7] and for Glc-I-P in the yeast enzyme [1,2].

Since the reaction proceeds in the presence of Pi alone, we conclude that the enzyme is acting on intracellular glycogen, which is present at a concentration of about 9%. This compares well with the level in the intact yeast wich is 10.5%. It appears that the immediate environment of the enzyme can be modified by altering the external environment, Thus, we feel that these permeabilized structures can be used for studies on some of the factors which control glycogen degradation in yeast [5].

We thank Dr David Santiago for his advice and Mr Felix Liard for his help on the photographic work. Supported by NIH Grant 2 T 01 AI00171-11 from the U.S. Public Health Service.

References

1 Saga rd i a , F . , G o t a y , I .C. a n d R o d r i g u e z , M. ( 1 9 7 1 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 42 , 8 2 9 - - 8 3 5

2 F o s s e t t , M., Muir , L.W., Nie lsen , L .D. a n d F i sche r , E .H. ( 1 9 7 1 ) B i o c h e m i s t r y 1 0 , 4 1 0 5 - - 4 1 1 3 3 S c h l e n k , F . a n d Z y d e k - C w i k , C .R. ( 1 9 7 0 ) Arch . B i o e h e m . B i o p h y s . 1 3 8 , 2 2 0 - - 2 2 5

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4 Adams, B.G. (1972) A n a l Biochem. 45, 137--146 5 Li~sch , M.H. and Preiss, J. (1967) BacterioL Proe. p. 110 6 Reeves, R.E. and Sols, A. (1973) Biochem. Biophys. Res. Commun. 50, 459--466 7 Sagardia, F. and Green, J.W. (1969) Biochim. Biophys. Acta 185, 80--87 8 Trevelyan, W.E. and Harrison, J.S. (1956) Biochcm. J. 63, 23---32 9 Dubois, M.K.A., Gilles, K.K., Hamilton, P.A. and Smith, F. (1956) Anal. Chem. 28, 350--357

10 Rodriguez, M. (1968) M.S. Thesis, University of Puerto Rico, Rio Piedras, Puerto Rico

The A u t h o r index of this vo lume can be found on p. 355.