novel down-stream effectors of small gtp-binding proteins of the ras-superfamily

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9TH INTERNATIONAL/6TH EUROPEAN JOINT SYMPOSIUM ON PURINE AND PYRIMIDINE METABOLISM IN MAN

Design and Methods The muscle biopsy showed a normal histol- ogy except for negative s ta in ing for MAD and a minimal residual MAD activity (0.43 U/g tissue). All exons of the AMPD1 gene were sequenced using genomic DNA. The muta t ions were confirmed in the cDNA of the biopsy. Results The pat ien t carries the known m u t a n t allele (C34-T and C143-T). In the second AMPD1 allele, a new muta t ion was found: G468-T (gln156-his). This muta t ion was not detected in 50 control DNA samples. Conclusion This is the first pa t ien t wi th pr imary MADD who is not homozygous for the C34-T mutat ion. The G468-T muta t ion causes MADD at least in combination wi th the C34-T m u t a n t allele. For proper diagnosis of MADD both muta t ions should be analyzed.

63 NOVEL DOWN-STREAM EFFECTORS OF SMALL GTP-BINDING PROTEINS OF THE RAS-SUPER- FAMILY H. Grunicke, I. Tinhofer, S. Giselbreeht, K. Maly and F. Uberall, Ins t i tu te of Med. Chem. & Biochemistry, Univer- sity of Innsbruck, Austr ia

Objectives Small G-proteins of the Ras superfamily are implicated in a var iety of in t racel lu lar signal recognition and signal t rans- duction processes. For p21 ra~ in part icular , several effector mol- ecules have been identified. In addit ion to the canonical Ras-Raf- 1-MEK-MAPkinase cascade, the Ras-Rac-l-RhoA pa thway wi th connects Ras to cytoskeletal organization and integr in signaling has gained special at tention. Molecular details of signal t rans- mission of the la t ter cascade are, however, still obscure. Data from or laboratory indicate t ha t the EGF-induced Ca2+-influx is abrogated by microinjection of a neutra l iz ing anti-Ras antibody, expression of a dominant negative (N17) Ras or microinjection of dominant negat ive Rac-1. EGF-induced Ca2+-influx was not found to be affected by a blockade of Raf-1. It is concluded, therefore, t ha t the EGF-induced Ca2+-infiux is mediated by a Ras-Rac-1 pathway. Expression of t ransforming Ras causes dra- matic morphological a l terat ions which include a disassembly of actin s tress fibers. Actin cytoskeleton organizat ion is also known to be under control of Rac-1 and Rho. Data will be presented demonst ra t ing t ha t expression of a dominant negative m u t a n t of atypical PKC lambda inhibi ts the Ras-induced disassembly of s tress fibers. This effect is specific for PKC lambda and not seen after expression of dominant negative m u t an t s of the epsilon- or alpha-isotypes of PKC. Fur thermore , a constitutively active mu- t an t of PKC lambda mimics t ransforming Ras, i.e. causes a disassembly of actin s tress fibers in the absence of oncogenic Ras. The da ta demonst ra te tha t the EGF-induced Ca2+-influx which had not been known to be Ras-dependent is mediated by a Ras-Rac-1 signal ing mechanism and tha t the Ras-induced reor- ganizat ion of actin cytoskeleton represents a PKC lambda-depen- dent process.

64 IDENTIFICATION OF THE SUBSTRATE BIND- ING AND CATALYTIC SITES OF THE 5-AMINO- IMIDAZOLE-CARBOXAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE ACTIVITY IN PurH Karen Gunn and G. Peter Beardsley

Objectives The objective of this project is to define the s tructure- function relat ionships of the AICARFT binding site in h P u r H with its subs t ra tes as an essential step in the process of s tructure- based computat ion-assis ted design of potent ial inhibi tors t ha t may be developed as folate antagonis t chemotherapeut ic agents.

Design and Methods We are current ly util izing two approaches to a t t a in our goal: 1) Affinity labeling with active-site directed irreversible inhibitors, 2) Site directed mutagenesis based on crystallographic insights. Results Prel iminary work with a 10-carboxaldehyde derivative folate analog has shown tha t i t completely inhibits AICARFT activity. Fur the r character izat ion of the complex is underway. Two of six potent ial mu tan t s have been constructed based on the puta t ive binding site of the AICARFT crystal structure: his t idine 266 to a lanine and lysine 265 to alanine. Prel iminary results show tha t ne i ther m u t a n t has AICARFT activity. The lysine to a lanine m u t a n t re ta ins the ability to bind to an AICAR sepharose column. Conclusion A 10-carboxaldehyde derivative folate analog is a potent ial affinity label for the AICARFT active site. Lysine 265 and hist idine 266 are potentially critical residues for the binding site and/or catalytic activity of AICARFT.

65 ADENOSINE RECEPTORS Gutensohn W., Ins t i tu te of Anthropology and Human Ge- netics, Universi ty of Munich, Goethestr, 31, D 80336 Mu- nich, Fed. Rep. Germany

Objectives As a short introduction to the "Adenosine Workshop" an overview on h u m a n adenosine receptors will be given. After clarifying nomenclature (P1- and P2-receptors) common features of as well as characterist ic differences between the 4 well de- scribed subtypes of h u m a n adenosine receptors (A1, A2A, A2B and A3) will be t reated on the genomic, s t ructura l and functional/ pharmacological level. St ructure function relat ionships are dis- cussed with respect to ligand binding and to coupling with the intracel lular signal t ransduct ion machinery (G-proteins). Regu- lation of expression and t issue distr ibut ion are demonst ra ted in establ ished examples of a physiological role of the receptor subtypes.

66 RENAL URATE HANDLING STATUS IN THE AB- NORMAL ANTIDIURETIC HORMONE SECRE- TION Tomoya Hamaguchi, Hiromu Nakajima, Koji Yamamoto, Koji Tomita, Makoto Moriwaki, Naoto Ito, Jun-ichiro Miya- gawa, Mitsuyoshi Namba, Toshiaki Hanafusa, Yuji Matsu- zawa, Osaka, J a p a n

Objectives To analyze the relative effects of ant idiuret ic hormone (ADH) and the extracellular fluid (ECF) volume on the dynamics of ur inary excretion of urate. Design and Methods The fractional u ra te excretion (FEvA) was surveyed in two pat ien ts with central diabetes insipidus (DI), and in one pat ient with syndrome of inappropriate ADH secretion (SIADH). Results The two cases with DI showed hyperuricemia (7.8 mg/dl, 8.2 mg/dl), and the SIADH case showed hypouricemia (2.4 mg/dl). At the end of the water-restr ict ion tes t in DI patients , ADH (5U) was injected subcutaneously. FEuA fell from 3.5% to 2.4% in one patient , and from 3.3% to 2.3% in the other. In the case of SIADH, however, FEuA was relatively higher (13.5%). During the treat- ment of SIADH by oral sal t replacement and water restriction, FEuA fell gradually to 6.3%. Conclusion 1. The ur inary excretion of ura te was a dominant factor in

de termining the serum ura te level. 2. FEuA was mainly affected by the ECF volume. 3. ADH played a role in modifying the regulation of ura te

excretion.

258 CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997

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