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

The study reported here aimed to purify a cysteine proteinase from neurula embryos of Xenopus laevis, since this enzyme was thought to be involved in yolk-lysis in developing embryos. The purification procedure consisted of fractionation of an embryonic extract by means of 30-90% ammonium sulfate, chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose, gel filtration on Sephadex G-75 and affinity chromatography on concanavalin A-agarose. The purified enzyme had a molecular weight of 30 kDa according to both SDS-PAGE and Sephadex G-75 gel-filtration and an optimum pH of 5.5, and it preferentially cleaved the synthetic substrate, Z-Phe-Arg-MCA. Its activity was inhibited by Z-Phe-Phe-CHN2, a specific cathepsin L inhibitor, as well as by leupeptin and E-64. The NH2-terminal amino acid sequence of the enzyme was similar to that of chicken cathepsin B. These characteristics indicate that the purified enzyme is a member of the cysteine proteinase family. The antibody raised against the purified enzyme specifically stained a 30 kDa protein of neurula embryo extracts on immunoblot tests. The enzyme effectively digested Xenopus yolk proteins when the NaCl concentration in test solutions was 0.2 M. It was also confirmed that cysteine proteinase inhibitors inhibited yolk-lysis by the enzyme.
 ...
PMID:Purification and properties of embryonic cysteine proteinase which participates in yolk-lysis of Xenopus laevis. 973 41

A cysteine proteinase inhibitor has been purified from immature fruit of Malus domestica (var. Royal Gala). The M(r) of this apple cystatin is estimated to be 10,700 by MALDI-TOF mass spectrometry, 11 300 by SDS-PAGE and 11,000 by gel filtration. It is a relatively strong inhibitor of papain with a Ki value of 0.21 nM and also inhibits ficin and bromelain but not cathepsin B. An amino acid sequence was obtained from a peptide produced by trypsin digestion of the inhibitor. Comparison with other plant sequences shows a high degree of homology with other phytocystatins. As the single cysteine proteinase inhibitor detectable in immature apple fruit (5-8 mm diameter), levels of 83.3 pmol/g FW were determined. In larger fruit (up to 16 mm diameter) significantly less inhibitor was present (6.9 pmol/g FW). Given these low levels, it is postulated that this inhibitor has an endogenous role in apple fruit development rather than one of protection against pest or microbial attack.
 ...
PMID:A cysteine proteinase inhibitor purified from apple fruit. 978 44

The lysosomal cysteine peptidase cathepsin B was found to be associated with plasma membrane/endosomal fractions of murine B16 amelanotic melanoma cells. Confocal microscopy with three dimensional image analysis indicated that cathepsin B was associated with the external basal cell surface, which would be consistent with its proposed role in degradation of extracellular matrix proteins. We purified and partially characterized cathepsin B from homogenates of murine liver and B16 amelanotic melanoma cells and from lysosomal and membrane/endosomal fractions of the B16 tumor cells. By SDS-PAGE under reducing conditions, the purified cathepsin B from the tumor homogenates was resolved as a single protein band of Mr 31000, corresponding to the single chain form of cathepsin B. In contrast, cathepsin B from liver homogenates was resolved as two bands of Mr 31000 and 24000, corresponding to the single chain and the heavy chain of the double chain form, respectively. The tumor cathepsin B consisted of four isozymes with pIs of 5.64, 5.33, 5.2 and 5.1, whereas the liver cathepsin B consisted of five isozymes with pIs of 5.64, 5.5, 5.45, 5.35 and 5.3. The additional acidic isoforms of cathepsin B in the B16 tumor probably reflect altered glycosylation in tumors. The commonality of isoforms in the B16 plasma membrane/endosomal and lysosomal fractions suggests that retrograde trafficking of cathepsin B from the lysosome to the endosome and its exocytotic release result in the association of cathepsin B with the tumor cell membrane.
 ...
PMID:Tumor cell membrane cathepsin B. 979 42

A new technique has been developed to identify active proteinases in endosomes that does not require prior isolation of organelles and extraction of the active enzymes. [125I]Iodotyrosylalanyldiazomethane was reversibly conjugated to transferrin to selectively deliver it to endosomes. The protein was conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enzymes were separated from the transferrin after SDS/PAGE under reducing conditions. Uptake of the protein-inhibitor conjugate and incorporation of inhibitor into cathepsins was blocked at 4 degreesC, demonstrating that the conjugate enters cells by receptor-mediated endocytosis. Furthermore, endocytosed transferrin-inhibitor conjugate could be recycled back to the extracellular medium and binding to the transferrin receptor could be blocked by native transferrin. Labelling of the enzymes was not blocked by incubating cells at 16 degreesC, consistent with the majority of the reagent being targeted to endosomes. The inhibited enzymes remained conjugated to transferrin, showing that the disulphide bond between the transferrin and inhibitor was not reduced in the endosome. Results from these studies show that endosomes contain both intermediate and late biosynthetic forms of active cathepsin B, which are indistinguishable from those found in mature lysosomes. These results indicate that the active enzymes in endosomes are not early biosynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.
 ...
PMID:Design of a transferrin-proteinase inhibitor conjugate to probe for active cysteine proteinases in endosomes. 984 79

A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses. In normal articular cartilage (n = 6 animals), significant amounts of enzyme were detected only in hypertrophic chondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n = 5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n = 6), but increased progressively during serial subculture, reaching a maximum at passage 5-6. In contrast, primary cultures of dyschondroplastic chondrocytes (n = 3) expressed abundant cathepsin B.
 ...
PMID:Immunolocalization of cathepsin B in equine dyschondroplastic articular cartilage. 988 87

Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are the key transcription factors for the regulation of the cellular cholesterol level. To identify proteolytic enzymes for SREBPs, a fluorogenic peptide substrate, MOCAc-GRSVLSFK(Dnp)rr-NH2, was synthesized according to the proposed cleavage site of human SREBP-2. In microsome fractions from hamster liver, we found a peptidase activity inhibitable by the synthetic inhibitor Ac-GRSVL-aldehyde with an IC50 of 40 nM. This peptidase separated into three peaks of approximately 400 kDa, 60 kDa, and 30 kDa (Mp400, Mp60 and Mp30 respectively) upon gel permeation chromatography. Mp30 was purified to apparent homogeneity with an Mr of 32 kDa. The partial amino acid sequence of Mp30 possessed homology to cathepsin B (EC 3.4.22.1). A 109 kDa protein band on SDS-PAGE which corresponded to Mp400 exhibited homology to neprilysin (EC 3.4.24.11) in partial amino acid sequence. These findings suggest several degradative pathways for SREBP in liver microsome membranes.
 ...
PMID:Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes. 1032 22

The objective of the study was to improve the understanding of the relationship between the effect of epinephrine plus exercise and meat tenderness. The calpain, calpastatin, and cathepsin B + L activities and postmortem proteolysis in porcine longissimus muscle were studied. The muscle glycogen stores were depleted in five pigs by s.c. injection of epinephrine (.3 mg/kg) at 15 h antemortem and exercise on a treadmill (5 min, 3.8 km/h) immediately before slaughter. Antemortem injection of epinephrine and treadmill exercise resulted in higher ultimate pH (6.32 vs 5.66 in control) and decreased (P < .05) thaw loss, cooking loss, and shear force values. The muscle energy depletion treatment increased (P < .05) the muscle mu-calpain activity measured 42 min postmortem, and at 24 h mu-calpain activity was still approximately 50% greater in the high ultimate pH group. Also, as the ratio of mu-calpain to calpastatin increased (P < .01), the overall proteolytic potential of the calpain system were greater. These observations suggest that the muscle energy level may influence the activity of the calpain system in the living animal. The high ultimate pH group showed lower (P < .001) cathepsin B + L activity in the myofibrillar and the soluble fractions after 8 d of storage, suggesting that the increased ultimate pH increased the stability of the lysosomal membrane and thereby reduced the release of cathepsins from the lysosomes during storage. The SDS-PAGE showed increased (P < .001) degradation of a 39-kDa band in the epinephrine and exercise-treated samples. Degradation products at 30, 31, and 32 kDa were labeled by troponin-T antibody in western blot. An appearing 24-kDa band was identified as a troponin-I degradation product in western blot. The proteolytic degradation pattern of myofibrillar proteins during storage differed in control and treated samples, supporting the hypothesis that calpain-mediated proteolysis was affected after treatment, resulting in meat with high ultimate pH.
 ...
PMID:Combined effect of epinephrine and exercise on calpain/calpastatin and cathepsin B and L activity in porcine longissimus muscle. 1049 49

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.
 ...
PMID:The membrane-bound basic carboxypeptidase from hog intestinal mucosa(1). 1051 94

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).
 ...
PMID:Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis. 1074 21

The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca2+-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of 14C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca2+-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain.
 ...
PMID:Inhibition of proteolysis by a cyclooxygenase inhibitor, indomethacin. 1107 71


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>