Carboxyl-terminal Amino Acid Residues Research Articles

Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max)

The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti(a). Protease K1 cleaves Ti(a) to Ti(a) (m), the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti(a). Protease K1 is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti(a) at pH 4. The characteristics of protease K1 are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti(a) at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti(a) to products other than Ti(a) (m). However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease G1 (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease G1 is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth.

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification, crystallisation and characterisation of carboxypeptidase from wheat bran

A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N-substituted dipeptides and tripeptides which contain l-proline. It had a pH optimum at pH 4·0 for Z-Glu-Tyr ( Z-benzyloxycarbonyl). The K m and k cat values for Z-Glu-Tyr at pH 4·0 and 30°C were 0·19 m m and 20 s −1, respectively. The enzyme hydrolysed Z-Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.

Effect of carboxypeptidase digestion of the human choriogonadotropin molecule on its thyrotropic activity.

Digestion of hCG by a mixture of carboxypeptidases B and Y results in an enzyme dose- and incubation time-dependent increase in its ability to stimulate the adenylate cyclase (AC) of human thyroid membranes. Treated under the same conditions [3 h at 37 C; enzyme to hCG ratio, 0.04 (wt/wt)], partially and highly purified hCG preparations display an increase of about 300% in thyroid AC-stimulating activity, while TSH displays a 30% decrease. In contrast, carboxypeptidase digestion of hCG under these conditions has no significant effect on its activity in the rat testis AC assay. The carboxypeptidase digestion results in cleavage of carboxyl-terminal amino acid residues 142--145 from the hCG beta-subunit; digestion of the hCG alpha-subunit is much less effective, as the carboxy-terminal amino acid residue 92 is removed from only about 13% of the hCG molecules. In accord with the results of amino acid analysis, a slight, if any, decrease in apparent molecular weight is found by gel filtration and polyacrylamide gel electrophoresis. In addition, carboxypeptidase digestion results in antigenic alterations of the molecule, as shown by a flatter slope of the dose-response curve in a hCG RIA and a 70% decrease in potency in a RIA that uses an antiserum to the hCG beta carboxy-terminal peptide. These data demonstrate that partial digestion of the hCG molecule with carboxypeptidase results in an increase in human thyroid AC-stimulating activity, with retention of the rat testis AC-stimulating activity.

The roles of carboxyl-terminal amino acid residues in higher plant ferredoxin.

The roles of carboxyl-terminal amino acid residues in higher plant ferredoxin.

The effect of elimination of amino acids from the carboxyl-terminal of the minor ribonuclease from Aspergillus saitoi by carboxypeptidase A on the enzymatic activity.

In order to investigate whether carboxyl-terminal amino acid is involved in the active site of a base non-specific ribonuclease from Asp. saitoi (RNase Ms), removal of carboxyl-terminal amino acid residues by digestion with carboxypeptidase A was investigated. By 24 hours digestion, about 3 serine residues and 1.0 alanine residue were removed from RNase Ms and its activity decreased to about 70% of the native enzyme so far as measured with ribonucleic acid (RNA) as a substrate. The pH optimum and Km of carboxypeptidase treated RNase Ms (CP-RNase Ms) were very similar to those of native RNase Ms so far as measured with RNA as a substrate. However, Km of CP-RNase Ms using ApC as a substrate seemed to be larger than that of native RNase Ms. The large increase in Km value was not observed in the early stage of digestion where about 3 serine residues were removed. The gross structure of CP-RNase Ms was quite similar to that of the native RNase Ms by judging from circular dichroism spectrum at wavelength between 230-205 nm. From the results described above, it was concluded that carboxyl-terminal amino acid was not involved directly in the active site of RNase Ms.

Letter: Carboxyl-terminal amino acid residue analysis of peptides. A new method.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCarboxyl-terminal amino acid residue analysis of peptides. New methodMarvin J. Miller and Gordon M. LoudonCite this: J. Am. Chem. Soc. 1975, 97, 18, 5295–5297Publication Date (Print):September 1, 1975Publication History Published online1 May 2002Published inissue 1 September 1975https://doi.org/10.1021/ja00851a055RIGHTS & PERMISSIONSArticle Views124Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (343 KB) Get e-Alerts Get e-Alerts

Studies on the influence of carboxyl-terminal amino acid residues on the activity and stability of human erythrocyte carbonic anhydrase B

Studies on the influence of carboxyl-terminal amino acid residues on the activity and stability of human erythrocyte carbonic anhydrase B

Purification and Characterization of a Dialyzable 0.6 S γ2-Globulin from Normal Human Plasma

Abstract A method is described for the isolation of a hitherto unknown class of dialyzable proteins from Cohn Fraction VI of normal human plasma. The isolated protein fraction, which was shown to contain primarily basic, γ2-, γ1-, and β-globulins, revealed on ultracentrifugation a single symmetrical refractive index gradient sedimenting with a coefficient of 0.6 S and was further distinguished by the absence of carbohydrate. Purification of one of its components, a γ2-globulin, was achieved by chromatography on DEAE-, triethylaminoethyl, and CM-celluloses in conjunction with diafiltration. The resulting preparation appeared monodisperse on paper and polyacrylamide gel electrophoresis at pH 8.9 and 4.3. The homogeneous γ2-globulin was characterized in terms of its major physicochemical and chemical properties. The molecular weight was determined to be 5100. This low molecular weight protein possesses a single polypeptide chain as deduced from its sole amino-terminal (phenylalanine) and carboxyl-terminal (lysine) amino acid residues. Moreover, this globulin is distinguished by its lack of histidine, arginine, tryptophan, methionine, and cysteine. Sequential Edman degradation established the first 6 residues as Phe-Gln-Val-Val-Asp-Tyr. Analysis of the circular dichroism spectrum of this protein showed the presence of approximately 60% β structure and 40% random coil. It should be pointed out that the techniques employed by certain investigators, namely the widespread usage of nonvolatile buffers, insufficient amounts of starting material, and dialysis have precluded up to now the isolation of low molecular weight plasma constituents especially since they are present in small amounts. Furthermore, while the role of these hitherto unknown plasma proteins has as yet not been established, it is believed that the discovery of this new class of plasma globulins deserves special attention in that the information gained from their study may prove to have considerable biochemical and physiological significance.

A bovine pancreatic enzyme catalyzing the conversion of proinsulin to insulin.

An enzyme that catalyzes the conversion of bovine proinsulin to insulin has been purified from a bovine pancreatic extract. The product of conversion was identified as insulin by aminoacid analysis and determination of carboxyl terminal aminoacid residues. The purified enzyme preparation showed one major protein band on polyacrylamide gel disc electrophoresis; it had a molecular weight of about 70,000 and an isoelectric point (pI) at a pH of 4.82.

Isolation and partial characterization of a carboxypeptidase from barley.

A carboxypeptidase has been purified from germinated barley. The preparations are homogeneous in ultracentrifugal analysis. In disc electrophoresis traces of impurities can be detected at high concentration. The purified enzyme liberates carboxyl‐terminal amino acid residues from a wide range of carbobenzoxy (Z)‐dipeptides. Peptides containing proline are not attacked. It does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. The enzyme has a pH optimum at pH 5.2, is inhibited by di‐isopropylphosphofluoridate and p‐chloromercuribenzoate, and has a molecular weight of about 90,000.

The Specific Mechanism of Activation of Human Plasminogen to Plasmin

Abstract The activation of human plasminogen in a 25% glycerol medium was carried out with low molar ratios of urokinase, streptokinase, trypsin, and pig heart activator. Starch gel electrophoresis in 8 m urea at pH 3.2 showed that each of the four plasmins possessed the same electrophoretic mobility. Upon reduction with 2-mercaptoethanol in 8 m urea, each plasmin was found to contain two major, electrophoretically unique components. Determination of the aminoterminal and the carboxyl-terminal amino acid residues of these plasmins indicated, in accordance with our previous studies, that the activation of human plasminogen proceeds primarily through the cleavage of a single arginyl-valine bond. Each of the plasmins contains two polypeptide chains connected by a disulfide bond.

Determination of the carboxyl-terminal amino acid residue of the β-galactosidase of Escherichia coli K 12

1. 1. The carboxyl-terminal amino acid residue of the β-galactosidase (EC 3.2.1.23) of Escherichia coli K 12 has been shown to be lysine by both hydrazinolysis and carboxypeptidase B (EC 3.4.2.2) digestion. One mole of lysine per mole of monomer was found on the basis of a monomer molecular weight of 135 000. 2. 2. Further evidence is presented suggesting that the monomer may be composed of more than one peptide chain and the suggestion is made that unusual covalent bonds may be present in the molecule.

Purification and chemical analysis of Shope papilloma virus

Rate zonal density gradient centrifugation of preparations of Shope papilloma virus revealed three components (slow, middle, and fast) which were morphologically and serologically related. The slow sedimenting component was noninfectious, contained little or no DNA, and had a buoyant density of 1.29 in CsCl. The middle and fast sedimenting components were infectious and had the same buoyant density of 1.34 in CsCl. The middle sedimenting component, yielding a single band upon equilibrium sedimentation in CsCl, was found upon analysis to contain protein and 12% DNA, but no essential lipid and no detectable polyamines. Protein end-group analyses by treatment with hydrazine or with carboxypeptidase A showed the carboxyl terminal amino acid residue to be threonine. The amino-terminal group was unreactive toward fluorodinitrobenzene. The viral protein was analyzed for total amino acid content. Infectious DNA was extracted from the virus by treatment with 80% phenol, and shown to be free of contaminating virus by equilibrium density gradient centrifugation in CsCl.

The peptide chains of some plant viruses

Tomato bushy stunt and potato X viruses and cucumber viruses 3 and 4 were analyzed for amino-terminal and carboxyl-terminal amino acid residues. No free amino-terminal groups could be detected for any of these viruses using the fluorodinitrobenzene and phenylisothiocyanate methods. Using a hydrazinolysis procedure, the C-terminal residues of tomato bushy stunt, potato X, and the cucumber viruses were found to be leucine, proline, and alanine, respectively. A subunit size was calculated for each virus from the number of moles of terminal amino acid found for a known weight of virus.

A new method for the degradation of peptides.

THE main problem in working out the structure of peptides is to establish their amino-acid sequence. An essential part of this task consists in determining the amino-terminal and carboxyl-terminal amino-acid residues of the peptide chain (the amino-acid and the amino-acid)1. Many methods are dealing with this problem2.