Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Naturally occurring disulphide-linked alpha s2- and kappa-casein in bovine milk were purified by gel chromatography on a column of Sepharose CL-6B. Four fractions (A-D) were obtained by elution with ammonium acetate-urea buffer. Fractions A and B, identified by SDS gel electrophoresis and amino acid sequence analysis, corresponded to disulphide-linked kappa-casein and alpha s2-casein respectively. Fraction C consisted of a mixture of alpha s1-, alpha s2-, and beta-casein. Separation of fraction C into its components was achieved by reversed-phase HPLC. The stability of the disulphide bridges in alpha s2- and kappa-casein was shown to differ with respect to reducing agents (dithioerythritol and 2-mercaptoethanol).
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PMID:Purification of disulphide-linked alpha s2- and kappa-casein from bovine milk. 185 53

Proteolytic activity of proteases associated with somatic cells isolated from high SCC milk and proteases associated with leukocytes isolated from bovine blood was assayed at pH 6.6 and 5.2 in a model system consisting of a beta-casein (.96%) substrate in Jenness/Koops buffer. Intact beta-casein and casein proteolysis products were measured by densitometric analysis of SDS-PAGE gels. Relative proteolytic activity was expressed as percentage degradation of beta-casein after 24 h at 37 degrees C per 1 x 10(6) cells/ml. Proteolytic activity associated with somatic cells isolated from bovine milk was 27.5 and 13.6% at pH 6.6 and 5.2, respectively. Proteolytic activity associated with leukocytes isolated from bovine blood was 16.0 and 8.4% at pH 6.6 and 5.2, respectively. Proteolytic activity at pH 6.6 was significantly higher for the somatic cells isolated from milk than for leukocytes isolated from blood. The reason for the difference in proteolytic activity of leukocytes isolated from blood of a healthy cow versus somatic cells isolated from milk produced by a cow with mastitis is not known. Further work is needed to determine whether the difference may be caused by a higher proportion of activated macrophages in somatic cells isolated from milk produced by cows with mastitis than in the leukocytes isolated from blood of healthy cows.
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PMID:Properties of proteases from milk somatic cells and blood leukocytes. 189 6

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.
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PMID:Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa. 189 32

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.
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PMID:Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta). 190 78

A novel endogenous inhibitor of the proteasome (high molecular weight multicatalytic protease) has been isolated and characterized from human erythrocytes. After purification by ion-exchange and sizing chromatography, the inhibitor displayed a native molecular mass of approximately 200 kDa and contained a single subunit of 50 kDa with an isoelectric point of 6.9. Although the inhibitor noncompetitively blocks proteolysis of [methyl-14C]-alpha-casein (Ki = 7.1 x 10(-8) M) and inhibits hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC, it did not affect hydrolysis of other peptide substrates, such as MeOSuc-Phe-Leu-Phe-MNA and Z-Ala-Arg-Arg-MNA. To further characterize the 50-kDa inhibitor, a monoclonal antibody (MI-8) was generated that showed specific binding upon Western blot analysis of both native PAGE and SDS-PAGE. Immunoprecipitation with MI-8 specifically removed inhibitor activity against the proteasome. The 50-kDa inhibitor is distinct from a previously described 40-kDa inhibitor of the proteasome (Murakami, K., & Etlinger, J.D. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7588-7592) on the basis of lack of cross-reactivity with MI-8 and dissimilar peptide digest patterns. It is suggested that these endogenous inhibitors may have a role in ATP/ubiquitin-dependent proteolysis and/or other cellular functions involving this protease.
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PMID:Isolation and characterization of a novel endogenous inhibitor of the proteasome. 191 59

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
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PMID:E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription. 192 88

A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.
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PMID:Purification and characterization of a fibrinogenase from Vipera lebetina (desert adder) venom. 192 82

A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.
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PMID:Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells. 199 64

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.
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PMID:Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase. 201 39

Inactivation of the plasma serine-proteinase inhibitor alpha 1-antitrypsin (alpha 1-AT) by neutrophil metalloproteinases has been reported [Vissers, George, Bathurst, Brennan & Winterbourn (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 1390a; (1988) J. Clin. Invest. 82, 706-711; Desrochers & Weiss (1988) J. Clin. Invest. 81, 1646-1650]. To identify the enzyme responsible, supernatant from neutrophils stimulated with phorbol 12-myristate 13-acetate was subjected to preparative SDS/PAGE, both with and without activation of latent metalloproteinases with HgCl2. The lanes were subsequently sliced into pieces, the slices incubated with equimolar amounts of type I collagen and alpha 1-AT in the presence of HgCl2, and the reaction products separated by SDS/PAGE. With the latent supernatant, the characteristic collagen-cleavage products and cleaved alpha 1-AT were present in the same slices, corresponding to an Mr of 80,000-85,000. On treatment with HgCl2 both degradative activities underwent the same molecular-mass shift to a position corresponding to Mr 60,000-65,000. Western blots of neutrophil supernatants, using a polyclonal antibody to purified collagenase, showed Mr values of 83,000 for the latent enzyme and 63,000 for the HgCl2-activated enzyme. Neutrophil collagenase was purified to homogeneity and shown also to exist in a second latent form with Mr 70,000. When activated to the Mr-63,000 form by HgCl2 and incubated with equimolar amounts of collagen and alpha 1-AT, collagenase cleaved alpha 1-AT at almost twice the rate at which collagen was cleaved. alpha 1-AT cleavage was inhibited by 1,10-phenanthroline and by high concentrations of collagen. That the purified collagenase did not contain a contaminant proteinase such as stromelysin was indicated by inability of the preparation to cleave casein. Taken together these results lead us to conclude that neutrophil collagenase is capable of degrading alpha 1-AT. Neutrophil gelatinase also cleaved alpha 1-AT, but cleavage was slow when compared with its activity against gelatin.
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PMID:Human neutrophil collagenase cleaves alpha 1-antitrypsin. 217 52


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