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A T Profy

Publications and source records attributed to A T Profy.

25 records · Page 2Linked to original sources
PubMed1989

Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.

The principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is located in the external envelope protein, gp120, and has previously been mapped to a 24-amino acid-long sequence (denoted RP135). We show here that deletion of this sequence renders the envelope unable to elicit neutralizing antibodies. In addition, using synthetic peptide fragments of RP135, we have mapped the neutralizing determinant to 8 amino acids and found that a peptide of this size elicits neutralizing antibodies. This sequence contains a central Gly-Pro-Gly that is generally conserved between different HIV-1 isolates and is flanked by amino acids that differ from isolate to isolate. Antibodies elicited by peptides from one isolate do not neutralize two different isolates, and a hybrid peptide, consisting of amino acid sequences from two isolates, elicits neutralizing antibodies to both isolates. By using a mixture of peptides of this domain or a mixture of such hybrid peptides the type-specificity of the neutralizing antibody response to this determinant can perhaps be overcome.

Amino Acid Sequence↗
PubMed1986

A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase.

Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups. We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme. We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit. Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon. The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme. A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro. These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation. The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein. However, the Cys395----Ala mutant is refractory to NEM. This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395. Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln). The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme. The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered. In these features, the mutant resembles the NEM-inactivated protein. We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.

Alanine↗
PubMed1984

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine.

The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40% enantiomeric excess of the L isomer was incorporated at the internal 2' site and the positions of equilibrium for the 2' in equilibrium with 3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2'(3') greater than internal 2' greater than 5', and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotected DL-alanine led to an excess of the D isomer at the internal 2' site, while reaction with the N-carboxy anhydride of DL-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.

Acylation↗
PubMed1983

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.

Alkaline Phosphatase↗