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C M Noyes

Publications and source records attributed to C M Noyes.

27 records · Page 2Linked to original sources
PubMed1984

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue.

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1983

Identification of the molecular defect in factor IX Chapel Hill: substitution of histidine for arginine at position 145.

Hemophilia B Chapel Hill is a mild hereditary hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. Previous studies have demonstrated that purified factor IX Chapel Hill has 8% of the activity of normal human factor IX and that the activation of factor IX Chapel Hill is defective in that only one of the two peptide bonds hydrolyzed during activation of normal factor IX is cleaved. The tryptic peptides from normal human factor IX and factor IX Chapel Hill were subjected to analysis by high-performance liquid chromatography. Comparison of the elution profile of the peptides obtained from factor IX Chapel Hill and normal factor IX demonstrated that the tripeptide Leu-Thr-Arg, which is derived from the normal molecule (positions 143-145) immediately amino-terminal from the Arg-Ala peptide bond at 145-146 that is cleaved during the activation of factor IX with factor XIa, was absent in the digest obtained from factor factor IX Chapel Hill. The elongated "activation peptide" from factor factor IX Chapel Hill was obtained by further high-performance liquid chromatographic fractionation and subjected to primary structure analysis. The following sequence, corresponding to positions 143-147, was obtained: Leu-Thr-His-Ala-Glu. Thus, the primary molecular defect in factor factor IX Chapel Hill is the substitution of histidine for arginine at position 145. This substitution precludes cleavage by factor XIa at this peptide bond, and the activation peptide region remains associated with the light chain of factor IXa Chapel Hill.

Amino Acid Sequence↗
PubMed1982

Influence of metal ions on prothrombin self-association. Demonstration of dimer formation by intermolecular cross-linking with dithiobis(succinimidylpropionate).

Interaction of certain metal ions with prothrombin and prothrombin fragment 1 has been shown to result in conformational change(s). Self-association of prothrombin and prothrombin fragment 1 in the presence of divalent cations has been reported. The present study has made use of a covalent cross-linking reagent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin in the presence of divalent cations. In the presence of certain divalent cations, prothrombin dimer is the product of such cross-linking. Optimal dimerization of bovine prothrombin requires preincubation with calcium ions prior to the addition of cross-linking reagent. Calcium ions are also required for dimerization after this preincubation period. A similar time dependence is not seen with human prothrombin. The dimerization of bovine prothrombin is also supported by strontium and gadolinium to an extent comparable to that of calcium and to a lesser extent by barium or manganese. Magnesium ions do not support dimerization. The results suggest that certain divalent cations either induce or stabilize a conformation of prothrombin which can self-associate to form dimers. The results further suggest that divalent cations are also necessary for the actual cross-linking process subsequent to this conformational change.

Amino Acid Sequence↗
PubMed1982

Cleavage and activation of human prothrombin by Echis carinatus venom.

The cleavage of human prothrombin by partially purified Echis carinatus venom (ECV) was investigated in the present report. Incubation of prothrombin with ECV resulted in the rapid cleavage of prothrombin to alpha-thrombin, with the release of fragment-1 and fragment-2. When dansyl arginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), a very effective inhibitor of thrombin, was included in the ECV-prothrombin solution, meizothrombin was rapidly formed. Only small amounts of meizothrombin-1 could be detected. Prolonged incubation (23 h) in the presence of DAPA, however, resulted in nearly quantitative conversion of meizothrombin to meizothrombin-1 and fragment-1. Kinetic studies strongly suggested that the conversion of meizothrombin to meizothrombin-1 was due to ECV and not meizothrombin autolysis. In addition, EDTA, which inhibits ECV, blocked the cleavage of meizothrombin. Amino terminal sequence analysis indicated that ECV cleaves human prothrombin at two sites; Gly158-Ser159 and Arg322-Ile323. The former site differs from the site of autolytic cleavage of meizothrombin which occurs at Arg155-Ser156. In contrast to reports in the literature, the results of the present study indicate that the release of fragment-1 does not precede activation of human prothrombin by ECV.

Amino Acid Sequence↗
PubMed1981

Characterization of the defect in activation of factor IX Chapel Hill by human factor XIa.

Factor IXChapel Hill (Factor IXCH), an abnormal Factor IX molecule isolated from the plasma of a patient with mild hemophilia B, has previously been shown to exhibit delayed activation by Factor XIa and calcium. In this study, we have found that Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation of clotting activity of Factor IXCH is approximately 60% of the rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN.

Amino Acid Sequence↗
PubMed1979

The covalent differences between bovine alpha- and beta-thrombin. A structural explanation for the changes in catalytic activity.

The partial covalent structure of bovine beta-thrombin has been determined by the use of automated Edman degradation and carboxypeptidase digestion of the component polypeptide chains separated by gel filtration following either reduction and carboxymethylation or performic acid oxidation. beta-Thrombin has been found to contain three peptide chains derived by proteolysis of the parent alpha-thrombin molecule. The A chain of alpha-thrombin has been cleaved at two points yielding a peptide (A1 chain) which contains 17 amino acids, beginning with threonine 14 and ending with lysine 30. The B chain of alpha-thrombin has been cleaved at two positions to yield a B1 chain which begins with the NH2-terminal isoleucine and terminates with lysine 65 and a B2 chain which begins with lysine 74 and continues through COOH-terminal serine 259. The A1 chain and B2 chain are linked by a disulfide bridge. Although there is no evidence for a covalent bond between the B1 chain and the B2-A1 chains, the B1 chain is tightly bound to the remainder of the molecule, for separation is achieved only under denaturing conditions.

Amino Acid Sequence↗
PubMed1977

Purification and partial characterization of deoxyribonuclease I from bovine parotid gland.

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNase's.

Amino Acids↗