Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01034 (cystatin C)
 3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
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PMID:Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. 348 17

The cathepsins B, H and L of human origin were isolated in pure form in sufficient quantities for structural characterization. The complete amino acid sequence of human liver cathepsin B was determined. Partial amino acid sequences of the human kidney cathepsin H and L show the highly conserved region around the active site cysteine. The cysteine proteinase inhibitors stefin A, human stefin B and human cystatin C were isolated, characterized and sequenced. Their amino acid sequences are compared with sequences of other protein inhibitors of the stefin and cystatin family, showing a high degree of homology throughout both families. The stefin and cystatin family, together with newly discovered kininogen family belong to the same superfamily of cystatins. The constructed dendrogram shows that the most closely related inhibitors so far sequenced are human stefin B and rat liver TPI.
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PMID:Human cysteine proteinases and their protein inhibitors stefins, cystatins and kininogens. 349 61

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.
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PMID:Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. 349 37

A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum.
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PMID:Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay. 351 Nov 54

Native gamma-trace, a small basic protein present in high concentration in cerebrospinal fluid, semen and neuroendocrine cells, but of unknown biological function, is shown to be a potent inhibitor of the cysteine proteinases papain, ficin, and human cathepsins B, H and L. It proves to be the tightest -binding protein inhibitor of cathepsin B so far discovered. The name cystatin C is proposed for gamma-trace to reflect the many similarities in activity and structure to chicken egg-white cystatin and mammalian cystatins A and B. The inhibition constants of cystatin C, taken together with its widespread distribution in human tissues and extracellular fluids, suggest that a physiological function could well be the regulation of cysteine proteinase activity.
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PMID:The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. 620 23

Procathepsin L, the precursor to a powerful lysosomal cysteine proteinase, has been purified to apparent homogeneity from guinea pig spermatozoa, a novel and previously unrecognized source of this catalytically active zymogen. In the range of pH 5.0, procathepsin L (39,000 M(r)) readily self-processed yielding a mature, single-chain proteinase (29,000 M(r)) and an intact propeptide (10,000 M(r)) by what appeared kinetically to be an intramolecular reaction mechanism. These characteristics resembled those reported for the "major excreted protein" (MEP) of malignantly transformed mouse fibroblasts-a protein that has been characterized as the precursor to the mouse analog of human cathepsin L (B. R. Troen, S. Gal, and M. M. Gottesman (1987) Biochem. J. 246, 731-735). Other characteristics shared by the guinea pig and mouse zymogens included proteolytic activity at pH 5.0, homologous N-terminal amino acid sequences, and immunological relatedness. It was thus concluded that acrosomal procathepsin L is the guinea pig analog of MEP. Acrosomal procathepsin L had a specific activity on benzyloxy-carbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) of 30 mumol min-1 mg-1 enzyme at pH 3.2 and 37 degrees C. Relative to the assay substrate, rates on other fluorogenic substrates were 90% for Z-Phe-Cit-NMec, 63% for Z-Leu-Leu-Arg-NMec, 43% for D-Phe-Ser(Bzl)-Phe-Phe-Ala-Ala-p-aminobenzoate (a "specific" cathepsin D assay substrate), and 32% for Z-Val-Val-Arg-NMec. No action was detected on Z-Arg-Arg-NMec or Arg-NMec. Mature cathepsin L showed the same relative order of substrate specificity as its proenzyme form, but the absolute rates were about 5-fold greater. Additionally, the mature (single-chain) form of cathepsin L displayed Km and kcat values on Z-Phe-Arg-NMec that yielded an exceptionally high catalytic coefficient (11,600 s-1 mM-1) compared to values reported for two-chain forms of cathepsin L. Self-processing by acrosomal procathepsin L at pH 5.5 was totally inhibited by leupeptin, cystatin C, Ep-475, and Z-Phe-Phe-CHN2 at 1 microM levels. Gossypol (0.1 mM) gave 94% inhibition. Interestingly, dextran sulfate (100 micrograms ml-1) gave a 3.6-fold increase in the rate of self-processing seen at pH 5.5--a phenomenon of potential physiological relevance in view of the high-negative-charge density present within the hyaluronic acid-rich outer layer (cumulus oophorus) of the ovum.
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PMID:Purification and characterization of procathepsin L, a self-processing zymogen of guinea pig spermatozoa that acts on a cathepsin D assay substrate. 748 6

A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA). 776 45

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.
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PMID:Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level. 801 75

Human cystatin D is a novel member of the cystatin superfamily of cysteine proteinase inhibitors present in saliva and tears. Two alleles of the cystatin D gene (CST5), encoding protein variants with either Cys or Arg as residue 26 in their 122-residue polypeptide chains, are present in the population. Expression of the two alleles was investigated by immunochemical analyses of the secreted cystatin D in saliva from individuals homozygous for each of the two alleles, with results demonstrating that both are expressed at similar levels. The inhibitory characteristics of the two cystatin D variants were studied, by determination of dissociation equilibrium constants (Ki) for their complexes with papain and with the mammalian cysteine proteinases, cathepsins B, H, L, and S. The results demonstrate that 1) cystatin D has a characteristic inhibition profile since it does not inhibit cathepsin B (Ki > 1 microM), and when compared to cystatin C and all other known cystatins it is a much poorer inhibitor of cathepsin L (mean Ki 25 nM) but binds cathepsin H and S relatively tightly (mean Ki values of 8.5 and 0.24 nM, respectively); and 2) the inhibitory activities of the two cystatin D variants are not significantly different, demonstrating that the presence of an extra cysteine residue in the cystatin D molecule affects neither the stability nor the functional activity of the inhibitor, thus explaining the widespread distribution of the Cys26-cystatin D encoding allele in the population. The inhibitory properties displayed by cystatin D suggest that it has a function in saliva as inhibitor of either endogenous or exogenous enzymes with cathepsin S- or H-like properties.
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PMID:Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases. 808 19

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37 degrees C, by approximately 60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates.
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PMID:Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy. 810 23


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