Stephen Oroszlan

Thin-layer chromatography of sub-nanomole amounts of phenylthiohydantoin (PTH) amino acids on polyamide sheets

Abstract Thin-layer chromatography of PTH amino acids on polyamide sheets with a fluorescent indicator gave a 50–100-fold increase in sensitivity when compared to thin-layer polyamide plates developed by spray techniques. Resolution of the individual residues in a standard mixture remains comparable to that described previously. Using this technique, the two-dimensional chromatography of each residue from automated sequence analysis is quite feasible even when small amounts of material are available.

Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins

The substrate specificity of the human immunodeficiency virus type 1 (HIV‐1) and type 2 (HIV‐2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag‐Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (K m and k m) obtained for the different substrates showed several hundred‐fold variation but were similar for the same substrate.

Translational suppression in retroviral gene expression.

Publisher Summary This chapter summarizes the present state of knowledge concerning translational suppression in retroviruses. Other viruses, using similar mechanisms, are mentioned only briefly and tangentially. Retroviruses are a unique class of viruses that have been found in all classes of vertebrates but not in other organisms. Perhaps, their most distinctive properties are the flow of information from RNA to DNA early in the infectious process, and the subsequent integration of the viral DNA into the chromosomal DNA of the host cell. Retroviruses are the causative agents of acquired immunodeficiency syndrome (AIDS) and of a variety of neoplastic diseases in man and domestic animals. Elements with striking similarities to retroviruses, termed retrotransposons, occur in yeast and many other eukaryotes; elements sharing some characteristics with retroviruses have also recently been observed in prokaryotes. Because of the apparent relationship between retroviruses and retrotransposons, this chapter discusses of retrotransposons as well as retroviruses. Though all retroviruses utilize translational suppression in pol-protein synthesis, different groups of retroviruses use two completely distinct types of translational suppression. One of these is in-frame or readthrough suppression and the other is ribosomal frameshifting.

Chromatographic analysis of the aminoacyl-trnas which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV

Abstract An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the “shifty” tRNAs proposed by Jacks et al. (Cell 55, 447–458, 1988) to carry out frameshifting may be hypomodified isoacceptors.

Substitution of proline with pipecolic acid at the scissile bond converts a peptide substrate of HIV proteinase into a selective inhibitor

The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC50 of approximately 1 microM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.

Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virys protease☆☆☆

The genetic locus and primary structure of the human immunodeficiency virus (HIV) protease was determined by comparing the data of protein analyses with the published data of the gene analysis. The complete sequence of HIV-1 and HIV-2 protease was synthesized by solid-phase peptide synthesis. The synthetic protease was capable of accurately cleaving synthetic peptide substrates corresponding to known cleavage sites in gag polyproteins of HIV-1, HIV-2, and murine leukemia virus. The chemical synthesis of protease confirms the DNA sequence and provides a means of rapidly producing active protease in substantial quantities for biochemical and physical studies.

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H‐Vat‐Ser‐Gin‐Asn‐Tyr*Pro‐He‐Val‐Gin‐NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV‐1 and HIV‐2 proteinases. Deletion of the terminal Val and Gin had only moderate effect on the substrate hydrolysis, while the deletion of the P4, Ser as well as P3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having a high frequency of occurrence in β turns resulted in good substrates, while large amino acids were unfavourable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV‐2 substrate binding pocket.

Separation of amino acid phenylthiohydantoins by high-performance liquid chromatography on phenylalkyl support

Abstract A high-performance liquid chromatographic analytical system is described for the separation, identification, and quantitation of phenylthiohydantoin (PTH) derivatives of all amino acids normally encountered in proteins. The system employs reverse-phase chromatography on a μBondapak phenylalkyl stationary phase. PTH derivatives of all amino acids can be identified and quantitated in a single 30-min analysis using linear gradient elution. The method can be applied equally well to the separation of methyl esters of the PTH-derivatized decarboxylic amino acids. The sensitivity of the method extends into the range of less than 10 pmol.

The effect of salt on the Michaelis Menten constant of the HIV-1 protease correlates with the Hofmeister series

The effect of different types of salt on the proteolytic activity of HIV‐1 protease was studied. At a similar ionic strength, the enzyme activity changed according to the salting out effect of the ions used (Hofmeister series). Kinetic studies showed that a stronger salting out effect of the ions rather than the higher ionic strength per se increased the affinity to the substrate (Km ) but in general did not alter the K cat value.

Hydroxylamine-stable covalent linkage of myristic acid in Goα, a guanine nucleotide-binding protein of bovine brain

G0 alpha, a guanine nucleotide-binding protein with a strong homology to the G1 alpha and Gs alpha regulatory proteins of adenylate cyclase, is shown to contain myristic acid. The attachment of myristate to the protein is stable to hydroxylamine treatment, and since the amino-terminal sequence of G0 alpha is typical of proteins with amino-terminal myristate, the inference is strong that G0 alpha is also myristylated at its amino-terminal glycine.