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Biomedical subjects

K Titani

Publications and source records attributed to K Titani.

At least 235 records · Page 13Linked to original sources
PubMed1983

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If.

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.

Amino Acid Sequence↗
PubMed1983

Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage of methionyl and tryptophanyl bonds. Comparison of the amino acid sequences of cathepsins B and H with each other and with that of papain demonstrates a striking homology among their primary structures. Sequence identity is extremely high in regions which, according to the three-dimensional structure of papain, constitute the catalytic site. The results not only reveal the first structural features of mammalian thiol endopeptidases but also provide insight into the evolutionary relationships among plant and mammalian thiol proteases.

Amino Acid Sequence↗
PubMed1983

Peptide antibody specific for the amino terminus of skeletal muscle alpha-actin.

The NH2-terminal peptide of skeletal muscle alpha-actin (S alpha N peptide), which contains a primary sequence unique to this actin isozyme, was used to prepare an isozyme-specific peptide antibody. S alpha N peptide was purified from chicken breast muscle actin by preparative reverse-phase HPLC and was coupled to hemocyanin. This complex was used to immunize rabbits in order to elicit actin antibodies specific for the skeletal muscle alpha-actin isozyme. The antibody obtained, called S alpha N antibody, was reactive with S alpha N peptide and with skeletal muscle alpha-actin as well as with cardiac muscle alpha-actin. S alpha N antibody did not react with either of the actin isozymes present in smooth muscle (smooth muscle alpha and gamma) or in brain (nonmuscle beta and gamma). S alpha N antibody was used to detect muscle-specific actin in differentiating mouse and human myoblasts by using immunoblots of myoblast extracts and immunofluorescent staining of fixed cells.

Actins↗
PubMed1982

Complete amino acid sequence of human brain calmodulin.

The complete amino acid sequence of calmodulin from human brain has been determined by using peptides derived from digests with trypsin. Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides were purified by means of reversed-phase high-performance liquid chromatography and analyzed with a sequenator. The protein contains 148 amino acid residues and has a molecular weight of 16792. As in other calmodulins, the amino-terminal residue of the protein is blocked by an acetyl group, and a trimethyllysine residue is located at position 115. The only difference between this sequence and those fully determined in other species is the assignment of amide groups.

Amino Acid Sequence↗
PubMed1982

Complete amino acid sequence of porcine heart citrate synthase.

The detailed proof of the 437-residue amino acid sequence (Mr 48,969) of porcine heart citrate synthase (EC 4.13.7) is described. The S-carboxymethylated protein has been cleaved at methionine (cyanogen bromide) and arginine (trypsin digest of citraconylated enzyme) residues to yield 14 and 17 major peptides, respectively. Peptides were initially fractionated by gel filtration, and those useful for sequence analysis were purified by high-performance liquid chromatography. Sequence analyses were performed on these primary peptides and on subpeptides generated by cleavage with the bromine adduct of 2-[(2-nitrophenyl)sulfenyl]-3-methylindole, Staphylococcus aureus V8 protease, trypsin, chymotrypsin, or acid. The overall sequence was confirmed by analyzing products of cleavage by hydroxylamine, acid, and subtilisin. A novel feature of the sequence is the identification of trimethyllysine at residue 368.

Amino Acid Sequence↗
PubMed1982

n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle.

The unusual NH2-terminal blocking group of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein was found to be amide-linked n-tetradecanoic acid by gas chromatographic-, direct chemical ionization-, and fast atom bombardment-mass spectrometry. In addition, fast atom bombardment mass spectrometry revealed the presence of an additional alanine which had been overlooked when the original sequence was determined. The corrected and completed NH2-terminal sequence of the 350-amino acid catalytic subunit is CH3(CH2)12CONH-Gly-Asn-Ala-Ala-Ala-Ala-Lys.

Amino Acid Sequence↗
PubMed1982

The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous.

Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology. This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure. The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP. This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP. The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP. One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence. As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.

Amino Acid Sequence↗
PubMed1982

Primary structure of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle.

The complete amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle is presented. Primary fragments for the sequence determination were obtained by limited proteolysis with various proteases or by cleavage with cyanogen bromide. The sequence of the 400 amino acid residues has two homologous regions, strongly suggesting tandem gene duplication. The predicted secondary structure suggests the presence of 42% alpha-helix, 23% beta-strand, and 23 beta-turns. The molecular weight of the subunit, as derived from the sequence, is 45,084 including a phosphate group at residue 95. This is significantly less than earlier estimates based on NaDodSO4 gel electrophoresis and sedimentation experiments. The structure is discussed in terms of putative sites of interaction with cAMP and with the catalytic subunit.

Amino Acid Sequence↗
PubMed1981

Human brain calmodulin: isolation, characterization, and sequence of a half-molecule fragment.

A Ca2+-binding protein from human brain has been purified to homogeneity and identified as residues 72-148 of calmodulin. This half-molecule fragment (CaM72-148) contains 11 of calmodulin's 15 basic amino acids (including one trimethyllysine) and demonstrates a higher isoelectric point. Both tyrosines and three of eight phenylalanine residues also occur in the fragment, giving rise to a somewhat different absorption spectrum. Though it contains two of calmodulin's Ca2+-binding sites, CaM72-148 binds only one Ca2+ per molecule with a dissociation constant of 17 microM. No biological activity, as judged by its inability to activate cyclic nucleotide phosphodiesterase, is observed. The sequence of amino acids is identical with that of residues 72-148 of bovine brain calmodulin [Kasai, H., Kato, Y., Isobe, T., Kawasaki, H., & Okuyama, T. (1980) Biomed. Res. 1, 248-264]. CaM72-148 is thought to arise through proteolysis, and its implications for the structure and physiological role of calmodulin are discussed.

Amino Acid Sequence↗
PubMed1981

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.

Amino Acid Sequence↗
PubMed1981

Primary structure of porcine heart citrate synthase.

The sequence of 437 amino acid residues of porcine heart citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO leads to acetyl-CoA), EC 4. 1. 3. 7] has been determined by the alignment of fragments generated by cleavage with cyanogen bromide and with trypsin. Isolation of the peptides was facilitated by recent developments in the high-performance liquid chromatography of peptide mixtures. The alignment of these peptides was consistent with that previously deduced from fragments derived by restricted cleavage of citrate synthase by limited proteolysis and cleavage of aspartyl-prolyl bonds and asparaginyl-glycyl bonds. The enzyme contains a modified amino acid, trimethyllysine, at residue 368, showing that the enzyme is subjected to post-translational modification.

Amino Acid Sequence↗
PubMed1981

Advances in protein sequencing.

Although this review cannot possibly cover the myriad of new ideas reported in the analyses of 1100 proteins, it does attempt to highlight procedures and strategies of interest, admittedly with a bias toward examples most familiar to us. It is clear that the scope, efficiency, and sensitivity of the analyses have increased and will continue to do so. Literally any protein sequence can now be solved. The diversity of tactics and strategies adds to the thrill of the chase. If the next few years continue to show the growing interplay between sequence chemists, X-ray crystallographers, and nucleic acid sequencing groups, we may truly begin to understand the meaning underlying the 20-letter hieroglyphics of protein chemistry.

Amino Acid Sequence↗
PubMed1981

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L.

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.

Amino Acid Sequence↗