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Biomedical subjects

K Titani

Publications and source records attributed to K Titani.

At least 253 records · Page 14Linked to original sources
PubMed1980

Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin.

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.

Amino Acid Sequence↗
PubMed1980

The structures of the carbohydrate moieties of bovine blood coagulation factor X.

Bovine blood coagulation factor X contains both asparagine-linked and threonine-linked oligosaccharides. The asparagine-linked chain is a mixture of a tridecasaccharide NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and a dodecasaccharide NeuAc alpha 2 leads to 6 Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and their partial desialylation products. The threonine-linked chain is a mixture of NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, NeuGly alpha 2 leads to 3Gal beta 1 leads to 3 (NeuAc alpha 2 leads to 6)GalNAc, and NeuGly alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, and their partial desialized forms. The carbohydrate moieties of the factor X subgroups, factors X1 and X2, are identical.

Animals↗
PubMed1980

Amino acid sequence of the light chain of bovine factor X1 (Stuart factor).

The detailed proof of the amino acid sequence of the 140 residues (16,193 daltons) of the light chain of bovine factor X1 (Stuart factor) is presented. Sequence analyses were performed on fragments obtained after chemical cleavage of asparagine-glycine and tryptophanyl peptide bonds and after various enzymatic digestion procedures. Twelve gamma-carboxyglutamyl residues are clustered in the amino-terminal 39 residues and 13 half-cystine residues are found in the carboxyl-terminal 91 residues, suggesting two domains in the light chain, one exceptionally anionic and the other extensively cross-linked by disulfides.

Amino Acid Sequence↗
PubMed1979

Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The sequences of two phosphopeptides isolated from the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase (type II) and from two of its cyanogen bromide fragments, have been determined. One phosphorylation site is a threonyl residue located approximately 180 residues from the blocked NH2 terminus. Its sequence is: -Gly-Arg-Thr-Trp-Thr(P)-Leu-Cys- and includes one of the three sulfhydryl groups present in the molecule. The second phosphorylated site within the sequence: -Val-Ser(P)-Ile-Asn- is located towards the carboxyl end of the protein where the other 2 cysteinyl residues also reside. The finding that phosphorylation of the catalytic subunit occurs on two discrete sites rather than at random suggests that it might be of physiological importance, e.g. in the regulation of enzyme activity.

Amino Acid Sequence↗
PubMed1979

Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

Amino Acid Sequence↗
PubMed1978

Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control.

The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.

Allosteric Regulation↗
PubMed1978

Amino acid sequence of two cyanogen bromide fragments of glycogen phosphorylase.

This communication presents the strategy and experimental details to prove the amino acid sequence of two large fragments of rabbit muscle glycogen phosphorylase generated by cleavage with cyanogen bromide. These fragments, CB18 and CB15, represent 241 of the 841 residues in the whole molecule. In addition to applying methods of automated liquid phase Edman degradation, techniques of selective immobilization and solid phase Edman degradation are used. One of the two cyanogen bromide fragments (CB15) contains two of the sites of cleavage with hydroxylamine which have proved to be important in the overall strategy of determining the complete sequence of this molecule. Together with the accompanying reports by Koide, A., et al., and Titani, K., et al. ((1978) Biochemistry 17 (first and third papers, respectively, in a series in this issue)), the present communication completes the proof of the amino acid sequence of phosphorylase and provides the basis for examining the relationship between its structure and function.

Amino Acid Sequence↗
PubMed1978

Sequence of the carboxyl-terminal 492 residues of rabbit muscle glycogen phosphorylase including the pyridoxal 5'-phosphate binding site.

This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined.

Amino Acid Sequence↗
PubMed1978

Covalent structure of a group-specific protease from rat small intestine. Appendix: crystallographic data for a group specific protease from rat intestine.

"Group-specific" protease (GSP) is a serine protease, obtained from rat small intestine, which preferentially inactivates the apo forms of certain pyridoxal phosphate requiring enzymes. The enzyme contains 224 amino acid residues in a single polypeptide chain and three disulfide bonds. In the present work the covalent structure has been determined and its homologous relationship to those of chymotrypsin, trypsin, and elastase has been established (approximately 33% identity with each). The residues forming the "charge-relay" system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in GSP, whereas an alanyl residue at position 176 of GSP corresponds to a residue which participates in the primary substrate binding site in serine proteases (Asp-177 in trypsin; Ser-189 in chymotrypsin). Three disulfide bonds in GSP occur in similar positions in chymotrypsin, trypsin, and elastase. However, GSP lacks a disulfide bond which is present in all known serine proteases (linking Cys-191 to Cys-220 in chymotrypsin). In view of the close proximity of this bond to both the primary and the antiparallel binding sites of various serine proteases, it is likely that its absence in GSP is related to the substrate specificity of this enzyme. It is concluded that GSP diverged from a common ancestor preceding chymotrypsin but following trypsin.

Amino Acid Sequence↗
PubMed1977

Complete amino acid sequence of rabbit muscle glycogen phosphorylase.

The sequence of the 841 amino acid residues in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy) which were fragmented by cleavage at methyonyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder of the sequence. The residues involved in the covalent and allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679, respectively.

Amino Acid Sequence↗
PubMed1975

Amino acid sequence of dogfish trypsin.

The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.

Amino Acid Sequence↗