Phosphorylation of serine residues 3, 6, 10, and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65alpha.

GAD65, the smaller isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an alpha/beta doublet of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase (GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in neurons and synaptic-like microvesicles in pancreatic beta-cells. Here we demonstrate that GAD65alpha but not beta is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the alpha/beta heterogeneity but represents a unique post-translational modification of GAD65alpha. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65alpha in different combinations of phosphorylated serines. Phosphorylation of GAD65alpha was located at serine residues 3, 6, 10, and 13, shown to be mediated by a membrane bound kinase, and distinguish the membrane anchored, and soluble forms of the enzyme. Phosphorylation status does not affect membrane anchoring of GAD65, nor its Km or Vmax for glutamate. The results are consistent with a model in which GAD65alpha and -beta constitute the two subunits of the native GAD65 dimer, only one of which, alpha, undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.

Mechanism of Agrobacterium beta-glucosidase: kinetic analysis of the role of noncovalent enzyme/substrate interactions.

The role of noncovalent interactions in the catalytic mechanism of the Agrobacterium faecalis beta-glucosidase was investigated by steady-state and pre-steady state kinetic analysis of the hydrolysis of a series of monosubstituted aryl glycosides, in which the hydroxyl groups on the glycone were substituted by hydrogen or fluorine. Contributions of each hydroxyl group to binding of these substrates at the ground state are relatively weak (interaction energies of 3.3 kJ/mol or smaller) but are much greater at the two transition states (glycosylation and deglycosylation). The strongest transition state interactions were at the 2 position (at least 18 and 22 kJ/mol for glycosylation and deglycosylation, respectively) with the interactions at the 3 and 6 positions contributing at least another 9 kJ/mol of binding energy at both transition states. The interaction at the 4 position is less crucial to transition state binding but important for stabilization of the glycosyl-enzyme intermediate. Comparison of observed rates with those for spontaneous hydrolysis of the same substrates provides evidence for oxocarbenium ion character at both transition states, that for deglycosylation apparently having the greater positive charge development at the anomeric center.

Substrate-induced inactivation of a crippled beta-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role.

The beta-glucosidase from Agrobacterium sp. catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358. Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme. Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue. Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis. Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme.

Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus.

The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic beta cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD65 in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD65 and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD65 molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD65 in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Th1 subset of CD4+ T cells and cytotoxic T cell responses.

Syntheses of 2-deoxy-2-fluoro mono- and oligo-saccharide glycosides from glycals and evaluation as glycosidase inhibitors.

Several fluorinated oligosaccharides, including 2-deoxy-2-fluoro derivatives of cellobiose, maltose, and maltotriose were synthesized by the action of fluorine or acetyl hypofluorite on the corresponding glycal peracetates. Temperature effects on the stereoselectivities of these reactions were examined. Addition of acetyl hypofluorite to several 2-substituted glycals in the gluco or galacto series gave 2,2-disubstituted arabino- or lyxo-hexose derivatives; 3,4,6-tri-O-acetyl-2-fluoro-D-glucal or the analogous galactal yielded 2-deoxy-2,2-difluoro arabino- or lyxo-hexose peracetates, whereas 2-acetoxy-3,4,6-tri-O-acetyl-D-glucal or the analogous galactal gave 2(R)-2-acetoxy-2-fluoro-arabino- or lyxo-hexose peracetates, respectively. 2-Acetamido-3,4,6-tri-O-acetyl-D-glucal gave 2(R)-2-acetamido-2-acetoxy-3,4,6-tri-O-acetyl-alpha-D-arabino-hexopyrano syl fluoride. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside was an inactivator of the exoglucanase from Cellulomonas fimi while 2-deoxy-2-fluoro-alpha-maltosyl and alpha-maltotriosyl fluorides were slow substrates of human pancreatic alpha-amylase and rabbit muscle glycogen debranching enzyme, respectively.

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.

Synthesis and anticonvulsant activity of 3-alkoxycarbonylaminomethylcarbonylamino-4-benzoylpyridines .

3-Alkoxycarbonylaminomethylcarbonylamino-4-(arylcarbonyl)pyr idines--in which the chlorophenyl ring of dipeptidylaminobenzophenones is replaced by a pyridyl ring--were synthesized and evaluated as anticonvulsants using subcutaneous pentylenetetrazole (scPTZ) and maximal electroshock (MES) induced seizure screening tests. The substituent on the aryl ring of the 4-arylcarbonyl moiety was a determinant of activity in both tests, the potency order of substituents being generally 2-F greater than 2-H greater than 2-Cl. Compounds possessing a 3-benzyloxycarbonylaminomethylcarbonylamino substituent exhibited moderate activity in the scPTZ test, whereas all 3-tert-butoxycarbonylaminomethylcarbonylamino derivatives were inactive. The test results in the scPTZ screen suggest that the 3-benzyloxycarbonylaminomethylcarbonyl(N-methyl)amino compounds may undergo biotransformation, at least in part, to pyrido[3,4-e]-1,4-diazepin-2-ones. 3-Alkoxycarbonylaminomethylcarbonyl(N-methyl)amino-substituted compounds were always more potent than analogous 3-alkoxycarbonylaminomethylcarbonylamino-substituted compounds in the scPTZ test, whereas they were equipotent in the MES screen. Following oral administration, 3-benzyloxycarbonylaminomethylcarbonyl(N-methyl)amino-4-(2-chlorob enzoyl) pyridine exhibited a potency greater than that of valproic acid but less than that of clonazepam in the rat scPTZ screening test. 3-Benzyloxycarbonylaminomethylcarbonylamino-4-(2-fluorobenzoyl)pyr idine was the most potent compound in the rat oral MES screening test, exhibiting an activity greater than that of clonazepam but less than that of phenytoin. The 3-alkoxycarbonylaminomethylcarbonylamino-4-(arylcarbonylpyridin es had moderate affinity for the benzodiazepine receptor site(s); the IC50s in displacing 10 nM [3H]flunitazepam were in the 0.37-15.11 microM range (clonazepam = 0.003 microM).