scholarly journals Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney

1987 ◽  
Vol 241 (1) ◽  
pp. 229-235 ◽  
Author(s):  
P E Butler ◽  
M J McKay ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Degradation Products ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Mouse Kidney ◽  
Amino Acid Residues ◽  
Peptide Bonds ◽  
Membrane Bound ◽  
Terminal Amino

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S–S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

scholarly journals Action of human liver cathepsin B on the oxidized insulin B chain

1983 ◽  
Vol 213 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M J McKay ◽  
M K Offermann ◽  
A J Barrett ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Cathepsin B ◽  
Human Liver ◽  
Degradation Products ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Cleavage Sites ◽  
Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

scholarly journals Purification and characterization of truncated ribonuclease inhibitor

1991 ◽  
Vol 275 (2) ◽  
pp. 541-543 ◽  
Author(s):  
J Hofsteenge ◽  
A Vincentini ◽  
S R Stone
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Surface Properties ◽  
Ribonuclease A ◽  
Amino Acid Residues ◽  
Ribonuclease Inhibitor ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino

A recombinant pig ribonuclease inhibitor (delta r-RI) lacking 90 or 93 N-terminal amino acid residues was isolated from a preparation of recombinant inhibitor. The kinetic parameters for the inhibition of ribonuclease A by delta r-RI were determined and found to be only slightly altered in comparison with the full-length inhibitor. The deletion did, however, affect the surface properties of RI. The results are discussed in relation to those obtained by Lee & Vallee [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883].

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

scholarly journals Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 633-639 ◽  
Author(s):  
Bryan J. Starkey ◽  
David Snary ◽  
Adrian Allen
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Amino Acid Residues ◽  
Gastric Mucus ◽  
Terminal Amino Acid ◽  
Terminal Amino

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×106) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×106). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×105for mucoprotein A and 2.8×104for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

Fractionation of hordein by preparative, continuous carrier-free electrophoresis

1968 ◽  
Vol 46 (10) ◽  
pp. 1301-1307 ◽  
Author(s):  
Ch. Ivanov ◽  
B. Mesrob ◽  
Z. Prusik
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Content ◽  
Amino Acid Content ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Basic Fraction ◽  
Acidic Amino Acids ◽  
Carrier Free ◽  
Terminal Amino

Barley hordein was fractionated by preparative, continuous carrier-free electrophoresis. Six fractions were obtained, one of which was in negligible quantity. Three of the fractions gave single symmetrical peaks. The amino acid content and the N-terminal amino acid residues of these fractions were determined. The ratios of basic to acidic amino acids showed that the fractions contained different protein substances. The most basic fraction, representing 10% of the total hordein, appeared to be pure since it contained only alanine as a N-terminal amino acid.

scholarly journals Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli

1994 ◽  
Vol 298 (1) ◽  
pp. 189-195 ◽  
Author(s):  
C Fraipont ◽  
M Adam ◽  
M Nguyen-Distèche ◽  
W Keck ◽  
J Van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Protein ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Operator Expression ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Laci Repressor

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

scholarly journals Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Kristian M. Silander ◽  
Päivi Pihlajamaa ◽  
Biswajyoti Sahu ◽  
Olli A. Jänne ◽  
Leif C. Andersson
Keyword(s):  
Amino Acid ◽  
Ornithine Decarboxylase ◽  
Amino Acid Sequence Identity ◽  
Regulatory Region ◽  
Mouse Kidney ◽  
Decarboxylase Activity ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Proximal Tubuli

We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.

PURIFICATION AND PARTIAL CHEMICAL CHARACTERIZATION OF SHEEP NEUROPHYSIN

1973 ◽  
Vol 74 (2) ◽  
pp. 226-236 ◽  
Author(s):  
Michel Chrétien ◽  
Claude Gilardeau
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Characterization ◽  
Terminal Amino Acid ◽  
Amino Acid Determination ◽  
Pituitary Glands ◽  
Terminal Amino ◽  
Partial Chemical ◽  
Acid Determination

ABSTRACT A protein isolated from ovine pituitary glands has been purified, and its homogeneity assessed by NH2- and COOH-terminal amino acid determination, ultracentrifugation studies, and polyacrylamide gel electrophoresis after carboxymethylation. Its chemical and immunochemical properties are closely similar to those of beef and pork neurophysins, less similar to those of human neurophysins. It contains no tryptophan (like other neurophysins) or histidine (like all except bovine neurophysin-I and human neurophysins). It has alanine at the NH2-terminus and valine at the COOH-terminus. Its amino acid composition is similar to, but not identical with those of porcine and bovine neurophysins.

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