Cathepsin D inhibitor from potato tubers (Solanum tuberosum L.).
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
M Baudys
Publications and source records attributed to M Baudys.
S-S bridges of cathepsin B and H from bovine spleen: a basis for cathepsin B model building and possible functional implications for discrimination between exo- and endopeptidase activities among cathepsins B, H and L.
Bovine spleen cathepsin B contains 7 disulfide bridges. Using different chemical and enzymatic cleavage methods we isolated fragments representing the individual disulfides: Cys14-Cys43, Cys26-Cys71, Cys62-Cys128, Cys63-Cys67, Cys100-Cys132, Cys108-Cys119, and Cys148-Cys252. A similar line of approach was applied to determine the S-S bridges of bovine spleen cathepsin H: Cys23-Cys66, Cys57-Cys99, Cys157-Cys207, and Cys212-Cys5A, where Cys5A is located in the propart portion of the procathepsin H chain. On the basis of the knowledge of the S-S bridges of cathepsin B a novel sequence alignment of papain and cathepsin B has been proposed. This enabled us to construct a reasonable 3D-model of cathepsin B and propose the region (a 18 residue insertion between Glu89 and Gly90 of papain) responsible for the carboxypeptidase activity of cathepsin B functioning as a "closure". A similar approach was applied to explain the aminopeptidase activity of cathepsin H. A general model of steric regulation of accessibility of the preformed "endopeptidase-like" binding cleft by distant parts of the polypeptide chain of the proteinases discussed is proposed as a factor determining the mode of binding and thus cleavage of polypeptide substrates.
Amino Acid Sequence↗
Disulfide bridges of bovine spleen cathepsin B.
Bovine spleen cathepsin B contains 7 disulfide bridges. Cleavage of the enzyme with cyanogen bromide gives rise to a large and a small fragment. The former contains all disulfide bridges. Their arrangement was determined by analysis of amino-acid sequences and compositions of subfragments prepared by cleavage of the large cyanogen-bromide fragment with trypsin, chymotrypsin and the staphylococcal proteinase using specific methods for the detection of S-S-bonds. Disulfide bridges link together Cys14-Cys43, Cys26-Cys71, Cys62-Cys128, Cys63-Cys67, Cys100-Cys132, Cys108-Cys119 and Cys148-Cys252.
Amino Acid Sequence↗
Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family.
A novel effective procedure for the purification of cathepsin D inhibitor from potatoes (PDI) was developed. The amino acid sequence of PDI was determined by analysis of the cyanogen bromide digest and of the limited tryptic and chymotryptic digest of the protein. The inhibitor is a single polypeptide chain protein consisting of 188 residues with a simple sugar moiety attached to Asn-19. The tentative disulfide pairings are also suggested. The sequence data clearly indicate that PDI is homologous with the soybean trypsin inhibitor (STI) (Kunitz) family. The active center of PDI for trypsin inhibition was identified as Pro-Val-Arg-Phe in analogy to STI.
Amino Acid Sequence↗
Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14.
The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.
Amino Acid Sequence↗
Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment.
The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.
Amino Acid Sequence↗
Amino acid sequence of bovine spleen cathepsin B.
The complete amino acid sequence of bovine spleen cathepsin B was determined by manual and automatic Edman degradation of fragments prepared by proteolytic or chemical digestion of the enzyme. The single-chain form of the enzyme consists of 253 amino acid residues and its Mr is 27,468 (carbohydrate moiety not included). The light chain (residues 1-47) and the heavy chain (residues 50-253) of the enzyme are linked by the sequence -Gly-Arg (residues 48 and 49) in the single-chain form. Bovine spleen cathepsin B shows 80% sequence homology with cathepsins B from other species. An outstanding feature of bovine spleen cathepsin B not observed with the other cathepsins B is the presence of two additional half-cystine residues.
Amino Acid Sequence↗
Comparison between prochymosin and pepsinogen from lamb and calf.
1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 2. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. Lamb prochymosin and pepsinogen are both glycosylated.
Amino Acid Sequence↗
Identification of the second (buried) cysteine residue and of the C-terminal disulfide bridge of bovine spleen cathepsin B.
Quantitative differences were found when bovine spleen cathepsin B was subjected to SH-group titration in the presence and in the absence of denaturing agents, as well as when the pH of the titration buffer was increased. The intra- and interchain thiol-disulfide exchange reactions accompanying the denaturation of cathepsin B were investigated by polyacrylamide gel electrophoresis in SDS and by gel filtration experiments. An identical behavior in these experiments showed also cathepsin B whose active site Cys29 only had been carboxymethylated; these findings suggested the presence of one additional SH-group. After conditions preventing thiol-disulfide exchange reactions, had been developed, the second SH-group (Cys240) was demonstrated independently in carboxymethylated cathepsin B by labeling with 4-(dimethylamino)azobenzene-4'-iodoacetamide and by selective isolation of the SH-peptide containing Cys240 on thiopropyl-Sepharose. As the second important result, a disulfide bridge formed by Cys148 and Cys252 in the C-terminal part of the chain was identified.
Amino Acid Sequence↗
Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen.
Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.
Amino Acid Sequence↗
Covalent structure of chicken pepsinogen.
Chicken pepsinogen is a glycoprotein consisting of a single polypeptide chain and containing the following 367 amino acid residues: Asp23, Asn16, Thr26, Ser41, Glu14, Gln11, Pro18, Gly31, Ala17, Cys7, Val25, Met9, Ile23, Leu28, Tyr22, Phe20, His8, Lys17, Arg7, Trp4. The Mr-value of the protein is 42 074. This value includes the carbohydrate moiety of the protein, i.e. Man3, (GlcNAc)7, (-SO3H)5. The primary fragmentation of the molecule was effected by limited trypsinolysis at arginine residues after preceding modification of the lysines with citraconic anhydride. All eight peptides expected in theory were obtained and their size, amino acid composition, and N-terminal amino acid sequence were characterized. To elucidate the amino acid sequence of these large fragments the latter were subjected to secondary cleavage by CNBr, trypsin (after removal of the protecting groups from the lysines), the proteinase from Staphylococcus aureus V8 strain, alpha-chymotrypsin, hydroxylamine, or dilute acid; the resulting peptides were isolated by gel permeation and ion-exchange chromatography and by the fingerprint techniques. Overlaps at sites of the arginine residues were obtained in an earlier study [Baudys, M. & Kostka, V. (1982) Collect. Czech. Chem. Commun. 47, 2814-2832]. Chicken pepsinogen shows the highest degree of homology with the primary structures of pepsinogens A. The internal homologies are apparent in the neighborhood of the two active aspartic acid residues. We have assigned tentatively chicken pepsinogen to the group of pepsinogens A (EC 3.4.23.1); this assignment is a result both of our sequence studies and of an investigation of the kinetic characteristics of the enzyme.
Amino Acid Sequence↗
Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay.
Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.
Animals↗
Amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins.
The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr- Pro-Gln-Asn-Gly-Asn-Gly-64His-Gly-Thr-His-68Cys-Ala- Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile- Ala-Gly-Thr-Ala-Pro-Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.
Amino Acid Sequence↗
Identification of the active site cysteine and of the disulfide bonds in the N-terminal part of the molecule of bovine spleen cathepsin B.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Amino acid sequence of the small cyanogen bromide peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins.
The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala-Thr-Pro-His-Val-Ala-Gly-Val-Ala-Gly-Leu-Leu-Ala-Ser-Gln-Gly-Arg-Ser-Ala-Ser -Asn-Ile-Arg-Ala-Ala-Ile-Glu-Asn-Thr-Ala-Asp-Lys-Ile-Ser-Gly-Thr-Gly-Thr-Tyr-Trp-Ala-Lys-Gly-Arg-Val-Asn-Ala-Tyr-Lys-Ala-Val-Gln-Tyr. The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C-terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements. The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit or error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.
Amino Acid Sequence↗
Glucose-induced release of glycosylpoly(ethylene glycol) insulin bound to a soluble conjugate of concanavalin A.
Treatment of diabetes mellitus by insulin injections provides long-term control of the disease but lacks any feedback response to glucose concentration changes, which finally leads to a number of life-threatening conditions. The purpose of this study was to improve and optimize an implantable, concanavalin A (Con A) based, glucose-responsive insulin delivery system studied earlier [Jeong, S. Y., Kim, S. W., Holmberg, D. L., and McRea, J. C. (1985) J. Controlled Release 2, 143-152], which can be used for long-term diabetes treatment. To optimize the "insulin component" of the delivery system, we prepared PheB1 insulin amino group monosubstituted monoglucosylpoly(ethylene glycol) (G-PEG) insulin conjugates (PEG M(r) 600 or 2000), which showed preserved bioactivity, significantly improved solubility and solution stability at neutral pH, and substantially suppressed hexamerization/dimerization. To improve the delivery system further, we synthesized and characterized a conjugate of Con A and monomethoxypoly(ethylene glycol) (mPEG, M(r) 5000) grafted hydrophilic poly(vinylpyrrolidone-co-acrylic acid) (PVPAA) with M(r) of 250,000. The optimal conjugate contained around eight PEG chains and two to three Con A tetramers attached through the amide bonds to the PVPAA chain. The Con A sugar binding characteristics were preserved, and, more importantly, Con A solubility at pH 7.4 substantially increased. This also holds true for a complex formed by the Con A conjugate and G-PEG insulin, which is soluble and does not precipitate under the physiologically relevant conditions under which the complex formed by the Con A conjugate and glycosyl insulin immediately precipitates. Finally, no leakage of the Con A conjugate from a membrane device was detected. Preliminary in vitro release experiments with Con A conjugate and G-PEG insulin complex enclosed in the membrane device showed a pulsative, reversible release pattern for G-PEG insulin in response to glucose challenges of 50-500 mg/dL, demonstrating the feasibility of the release system for use in planned, chronic in vivo studies with diabetic (pancreatectomized) dogs.
Acrylic Resins↗
Extending insulin action in vivo by conjugation to carboxymethyl dextran.
The biochemical and pharmacological properties of bioactive peptides and proteins can be altered by conjugation with polymers. This report describes site-specific attachment of insulin to activated carboxyl groups of carboxymethyl dextran (CMD, MW=51000) through the GlyA1 insulin amino group. On average, three or four insulin molecules were grafted to a CMD linear chain. Coupled insulin molecules were properly folded, and the bioactivity of conjugated insulin in the blood glucose depression assay was 9.6 IU/mg, which was only 2.6 times less than that for native insulin. The cell growth study indicated that the CMD-insulin conjugate was as mitogenic as insulin on vascular smooth muscle cells, whereas the starting CMD polymer was not. The insulin receptor binding constant of the conjugate (3.6 x 10[9] M[-1]) compared well with that of native insulin (7.6 x 10[9] M[-1]), indicating that the CMD chain does not present any major constraints to binding. Plasma clearance of CMD-insulin obeyed a two-compartment pharmacokinetic (PK) model with a CMD-insulin conjugate plasma elimination half-life of 114.1 min, which was significantly longer than that of soluble Zn-insulin (12.4 min). In contrast, pharmacodynamic (PD) profiles (blood glucose lowering effects) after intravenous (iv) administration of the conjugate or insulin in rats were not different. Subcutaneous (sc) administration of the conjugate resulted in a significantly prolonged plasma profile with a noncompartmental PK parameter mean residence time (MRT) of 103.5 min which was significantly longer than that of soluble Zn-insulin (40.5 min). This was reflected in the protracted PD effect of sc administered conjugate with time needed to reach minimum glucose concentration Tnadir of 95.7 min, which was significantly longer than that of insulin (62 min). We conclude that the conjugation of insulin to CMD leads to a bioactive conjugate with a delayed sc PD profile showing prolonged response, resembling intermediate acting insulin preparations.
Animals↗
Synthesis and characterization of poly(ethylene glycol)-insulin conjugates.
Human insulin was modified by covalent attachment of short-chain (750 and 2000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-PheB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG(2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were performed using analytical ultracentrifugation to quantitatively determine the association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dimers while Zn(2+)-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This independent method qualitatively suggests that mPEG-insulin conjugates behave similarly to Zn-free insulin in the concentration range studied and complements results from ultracentrifugation studies. The physical stability/resistance to fibrillation of mPEG-insulin conjugates in aqueous solution were assessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates are substantially more stable than controls but the mPEG(750 and 2000)-LysB29-insulin conjugates were only slightly more stable than commercially available preparations. Circular dichroism studies done in the far ultraviolet region confirm insulin's tertiary structure in aqueous solution is essentially conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal that there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000) decreased the bioactivity of the conjugates to about 85% of Lilly's HumulinR formulation. The characterization presented in this paper provides strong testimony to the fact that attachment of mPEG to specific amino acid residues of insulin's B-chain improves the conjugates' physical stability without appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.
Animals↗