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Yakovlev AA, Kvichansky AA, Lyzhin AA, Khaspekov LG, Gulyaeva NV. Glutamate treatment and preconditioning differently affect cathepsin B release and intracellular proteases in primary cultures of cerebellar granular cells. NEUROCHEM J+ 2013. [DOI: 10.1134/s1819712413020098] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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2
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TENO NAOKI, TSUBOI SATOSHI, OKADA YOSHIO, ITOH NORIO, OKAMOTO HIROSHI. Amino acids and peptides. ACTA ACUST UNITED AC 2009. [DOI: 10.1111/j.1399-3011.1987.tb03315.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
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3
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Nagy L, Nagata M, Szabo S. Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: Possible new targets of pharmacologic agents. World J Gastroenterol 2007; 13:2053-60. [PMID: 17465447 PMCID: PMC4319124 DOI: 10.3748/wjg.v13.i14.2053] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate.
METHODS: Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver.
RESULTS: Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations.
CONCLUSION: Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.
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Affiliation(s)
- Lajos Nagy
- Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
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4
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Duffy JM, Walker B, Guthrie D, Grimshaw J, McNally G, Grimshaw JT, Spedding PL, Mollan RA. The detection, quantification and partial characterisation of cathepsin B-like activity in human pathological synovial fluids. EUROPEAN JOURNAL OF CLINICAL CHEMISTRY AND CLINICAL BIOCHEMISTRY : JOURNAL OF THE FORUM OF EUROPEAN CLINICAL CHEMISTRY SOCIETIES 1994; 32:429-34. [PMID: 7918840 DOI: 10.1515/cclm.1994.32.6.429] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
In this study, the levels of the cysteine proteinase--cathepsin B were measured in diseased synovial fluids using a steady state fluorometric assay. Cathepsin B-like activity was shown to be present in all the samples analysed, with the rheumatoid arthritic synovial fluids possessing significantly higher concentrations (mean value ca. 416 mg/l) than the osteoarthritic fluids (mean value ca. 142.4 mg/l). In addition, upon treatment with pepsin, all of the rheumatoid arthritis samples were shown to possess additional cathepsin B-like activity, suggesting the presence of a reservoir of latent precursor molecules. By utilising a recently developed biotinylated affinity label for cathepsin B-like proteinases and sheep anti-(human cathepsin B) antibodies, used in combination with SDS-PAGE and Western blotting, the rheumatoid arthritic synovial cathepsin B was shown to exist in two forms with apparent molecular masses of M(r) 29,000 and 42,000. We propose that the former is a functionally active proteinase, whereas the latter is a pepsin activatable proform which, when cleaved by this aspartyl proteinase, is converted into a catalytically competent species of M(r) 20,000.
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Affiliation(s)
- J M Duffy
- Queen's University of Belfast, Department of Orthopaedic Surgery, Musgrave Park Hospital, Northern Ireland
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Martel-Pelletier J, Cloutier JM, Pelletier JP. Cathepsin B and cysteine protease inhibitors in human osteoarthritis. J Orthop Res 1990; 8:336-44. [PMID: 2324852 DOI: 10.1002/jor.1100080305] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The aim of this study was to determine the involvement of cathepsin B and its inhibitors in the proteolytic degradation of human osteoarthritic (OA) tissue. The characteristics of the cathepsin B found in both normal and OA cartilage and synovium were similar to those of the lysosomal cathepsin B. Two inhibitors of cysteine proteases were found with a molecular weight of 67,000 and 16,000 Da. The cartilage cathepsin B level of OA specimens (54.8 +/- 7.3 units/micrograms of DNA) was greater than the controls (39.8 +/- 3.2 units/micrograms of DNA). Mild-moderate graded samples (78.1 +/- 12.0 units/micrograms of DNA) had significantly higher levels of enzyme activity than the severely graded ones (31.4 +/- 3.9 units/micrograms of DNA, p less than 0.001) and controls (p less than 0.01). Compared to controls (2.3 +/- 0.4 units/mg of tissue w.w.), cysteine protease inhibitory activity in OA cartilage was decreased in specimens with severe lesions (1.5 +/- 0.2 units/mg of tissue). This was particularly noted in patients who had not received steroid injections (1.2 +/- 0.3 units/mg of tissue, p less than 0.05). In OA synovia, the cathepsin B level was greater (40.7 +/- 7.4 units/mg of tissue w.w., p less than 0.02) than in the controls (13.6 +/- 3.7 units/mg of tissue). The cysteine protease inhibitory activity was similar in OA synovium (1.7 +/- 0.2 units/mg of tissue w.w.) and in controls (1.5 +/- 0.3 units/mg of tissue). This data demonstrated an imbalance between the levels of cathepsin B and cysteine protease inhibitors in OA tissue. A decrease of specific inhibitors could be an important contributing factor, particularly in more severe lesions.
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6
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Watanabe M, Higashi T, Watanabe A, Osawa T, Sato Y, Kimura Y, Tominaga S, Hashimoto N, Yoshida Y, Morimoto S. Cathepsin B and L activities in gastric cancer tissue: correlation with histological findings. BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 1989; 42:21-9. [PMID: 2775560 DOI: 10.1016/0885-4505(89)90037-6] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Cathepsin B and L activities in cancerous and noncancerous mucosal tissues from 29 patients with gastric cancer were determined with a small amount of tissue homogenate. Both enzyme activities were significantly higher in cancerous tissues than in noncancerous tissues. The cathepsin B activity was higher with decreasing differentiation of the cancerous tissues, and also with increasing depth of invasion and metastasis to regional lymph nodes. Significantly high cathepsin B activity was observed in specimens of poorly differentiated adenocarcinomas, as well as in specimens from patients with extensive metastasis to n2 or n3 lymph nodes. These results suggest that high cathepsin B activity is characteristic of gastric cancer which invades and metastasizes. Therefore, in cases of marked elevation of cathepsin B activity in cancerous tissues, relatively extensive resection may be necessary to obtain a cure.
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Affiliation(s)
- M Watanabe
- First Department of Internal Medicine, Okayama University Medical School, Japan
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7
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Fujii H, Aratake H, Deng Li Rong, Nakamura M, Kawaguchi Y, Sakaguchi B. Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor A (Ict-A) gene from the larval hemolymph of the silkworm, Bombyx mori. ACTA ACUST UNITED AC 1989. [DOI: 10.1016/0305-0491(89)90026-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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8
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Cole TC, Melrose J, Ghosh P. Cysteine proteinase inhibitors of the canine intervertebral disc. BIOCHIMICA ET BIOPHYSICA ACTA 1988; 952:201-7. [PMID: 3257394 DOI: 10.1016/0167-4838(88)90116-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Several species of cysteine proteinase inhibitors have been demonstrated in the greyhound intervertebral disc which were resolved into four species (Mr 15,800, 16,600, 17,200 and 17,800) by gelatin-SDS-polyacrylamide gel electrophoresis. Reductive alkylation did not affect their inhibitory capability nor their electrophoretic migration on gelatin-SDS-polyacrylamide gel electrophoresis. The cysteine proteinase inhibitors from the nucleus pulposus and annulus fibrosus were identical as assessed by the aforementioned criteria, although the level in the nucleus was found to be higher than that in the annulus. Ion-exchange chromatography demonstrated distinct acidic and basic forms of the disc cysteine proteinase inhibitor. The latter species was the most abundant and its Mr was determined to be 16,900 by gelatin-SDS-polyacrylamide gel electrophoresis. Both forms were shown to be strongly inhibitory against the cysteine proteinases, papain and ficin, but were less strongly inhibitory against cathepsin B (EC 3.4.22.1). Presumably these disc cysteine proteinase inhibitors play a regulatory role in the metabolism of proteoglycans and collagen by endogenous cysteine proteinases.
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Affiliation(s)
- T C Cole
- Raymond Purves Research Laboratories, University of Sydney, Royal North Shore Hospital, St. Leonards, Australia
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9
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Brocklehurst K, Willenbrock F, Salih E. Chapter 2 Cysteine proteinases. ACTA ACUST UNITED AC 1987. [DOI: 10.1016/s0167-7306(09)60016-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/16/2023]
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10
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Tani Y, Ohkubo I, Higashiyama S, Kunimatsu M, Sasaki M. Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. B, COMPARATIVE BIOCHEMISTRY 1987; 88:429-41. [PMID: 3123124 DOI: 10.1016/0305-0491(87)90323-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.
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Affiliation(s)
- Y Tani
- Department of Biochemistry, Nagoya City University Medical School, Japan
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11
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Baici A, Knöpfel M. Cysteine proteinases produced by cultured rabbit V2 carcinoma cells and rabbit skin fibroblasts. Int J Cancer 1986; 38:753-61. [PMID: 3095250 DOI: 10.1002/ijc.2910380520] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Rabbit V2 carcinoma cells and normal rabbit skin fibroblasts produced cysteine proteinases with properties similar to those of purified rabbit liver cathepsin B. Both cell types secreted into the culture medium enzymes with an apparent Mr of 43,000, which reacted with synthetic substrates commonly used for cathepsin B. After limited proteolysis with pepsin or treatment at pH 3, the Mr = 43,000 species could be converted into forms with Mr = 34,000 and an increased specific activity. In the intracellular pool of both V2 carcinoma cells and fibroblasts, a cysteine proteinase with the same Mr of cathepsin B (27,000) was found. Despite the similarity in molecular size, substrate specificity and sensitivity to inhibitors, the tumor and fibroblast enzymes were not identical in their stability at pH greater than or equal to 7 and were produced by the 2 cell types in considerably different amounts. In terms of enzyme units and normalized to an equal cell number, the ratios of fibroblast enzyme/tumor enzyme were as follows: secreted 130-150; intracellular, 150-180. The pH stability of the cysteine proteinases was determined quantitatively by measuring the half-life of enzyme activity. At pH 8.0 and 25 degrees C the secreted tumor cysteine proteinase had a half-life of at least 5 hr, whereas the secreted fibroblast enzyme and liver cathepsin B had half-lives of 8.8 min and 4.4 min, respectively.
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12
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Joronen I, Hopsu-Havu VK, Manninen M, Rinne A, Järvinen M, Halonen P. Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay. J Immunol Methods 1986; 86:243-7. [PMID: 3511154 DOI: 10.1016/0022-1759(86)90460-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
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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13
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Bige L, Ouali A, Valin C. Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle. BIOCHIMICA ET BIOPHYSICA ACTA 1985; 843:269-75. [PMID: 3877527 DOI: 10.1016/0304-4165(85)90148-5] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
A low-Mr tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their Mr of about 14 000, their stability with heating at 80 degrees C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (Ki) were determined for these three cysteine proteinases but for cathepsin H, association (kass) and dissociation (kdiss) rate constants were also evaluated. Ki values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, Ki was in the range of 0.1-1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-Mr protein inhibitor in controlling 'in vivo' cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.
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14
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Abstract
When human placental extract was chromatographed on a Sephadex G-75 column, cysteine proteinase inhibitors with molecular weights of 80 000 and 12 300 were eluted. The high molecular weight peak (CPI-H) was identified as alpha-cysteine proteinase inhibitor. The thermostable low molecular weight peak (CPI-L) inhibited plant proteinases (papain, ficin and bromelain) as well as cathepsins B, H and L isolated from the human placenta. No cross-reactivity was observed between placental CPI-L and serum alpha-CPI.
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15
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Hirado M, Tsunasawa S, Sakiyama F, Niinobe M, Fujii S. Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor. FEBS Lett 1985; 186:41-5. [PMID: 3891407 DOI: 10.1016/0014-5793(85)81335-1] [Citation(s) in RCA: 57] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin.
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16
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Purification and characterization of an inhibitor of the cysteine protease from the hemolymph of Sarcophaga peregrina larvae. J Biol Chem 1985. [DOI: 10.1016/s0021-9258(18)89186-4] [Citation(s) in RCA: 33] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
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17
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Hopsu-Havu VK, Joronen I, Havu S, Rinne A, Järvinen M, Forsström J. Serum cysteine proteinase inhibitors with special reference to kidney failure. Scand J Clin Lab Invest 1985; 45:11-6. [PMID: 3919439 DOI: 10.3109/00365518509160966] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Serum levels of proteins reactive in radioimmunoassay with an antiserum prepared in rabbits against purified human spleen neutral cysteine proteinase inhibitor (NCPI) was determined in 70 healthy controls and from 80 patients suffering from suspected or proven kidney failure. The values varied from less than 0.2 mg/l in normal sera to levels over 2 mg/l in some patient sera. Serum level of NCPI was found to roughly correlate with serum creatinine values. However, there were sera with high NCPI levels which did not have increased serum creatinine values. In sera with high NCPI levels subjected to double radial immunodiffusion two precipitin lines, one completely and the other partially identical to NCPI were registered. After fractionating of serum proteins with gel chromatography on Sephadex G 100, two protein peaks of immunological similarity to purified NCPI were found: one low molecular weight (MW around 12,000) and one high molecular weight (MW around 100,000). The low molecular weight NCPI-like material appeared to inhibit human cathepsin B and papain and is thus free serum NCPI. alpha-Cysteine proteinase inhibitor did not increase with serum creatinine as did NCPI.
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18
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Isolation of a Sputum Cysteine Protease Inhibitor. ACTA ACUST UNITED AC 1985. [DOI: 10.1016/b978-0-08-031739-7.50036-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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19
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Katunuma N, Kominami E. Molecular basis of intracellular regulation of thiol proteinase inhibitors. CURRENT TOPICS IN CELLULAR REGULATION 1985; 27:345-60. [PMID: 3912118 DOI: 10.1016/b978-0-12-152827-0.50037-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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20
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Rejmanová P, Kopecek J, Duncan R, Lloyd JB. Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers. Biomaterials 1985; 6:45-8. [PMID: 3971018 DOI: 10.1016/0142-9612(85)90037-7] [Citation(s) in RCA: 105] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in rho-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of rho-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of rho-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Gly-Phe-NAp where 5.1% of the bound rho-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.
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Ohkubo I, Kurachi K, Takasawa T, Shiokawa H, Sasaki M. Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry 1984; 23:5691-7. [PMID: 6441591 DOI: 10.1021/bi00319a005] [Citation(s) in RCA: 169] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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22
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Liao JC, Lenney JF. Cathepsins J and K: high molecular weight cysteine proteinases from human tissues. Biochem Biophys Res Commun 1984; 124:909-16. [PMID: 6439198 DOI: 10.1016/0006-291x(84)91044-1] [Citation(s) in RCA: 28] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.
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23
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Pagano M, Esnard F, Engler R, Gauthier F. Inhibition of human liver cathepsin L by alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine proteinase inhibitor from human serum. Biochem J 1984; 220:147-55. [PMID: 6547604 PMCID: PMC1153604 DOI: 10.1042/bj2200147] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.
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24
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Davies ME, Barrett AJ. Immunolocalization of human cystatins in neutrophils and lymphocytes. HISTOCHEMISTRY 1984; 80:373-7. [PMID: 6429090 DOI: 10.1007/bf00495420] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The endogenous inhibitors of cysteine proteinases, cystatin A and cystatin B, have been localized in sections of human liver by indirect immunofluorescence microscopy. Cystatin A was localized in neutrophils and cystatin B was found in lymphocytes. Both types of cell were present in the blood vessels and within the sinusoids between the parenchymal cells. Cystatin A was also found in some of the parenchymal cells surrounding the blood vessels but, in contrast to the uniform fluorescence of the leucocytes, the staining in the parenchymal cells was granular and rather irregular.
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25
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Barrett AJ, Davies ME, Grubb A. The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochem Biophys Res Commun 1984; 120:631-6. [PMID: 6203523 DOI: 10.1016/0006-291x(84)91302-0] [Citation(s) in RCA: 215] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
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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Szego CM, Pietras RJ. Lysosomal functions in cellular activation: propagation of the actions of hormones and other effectors. INTERNATIONAL REVIEW OF CYTOLOGY 1984; 88:1-302. [PMID: 6145684 DOI: 10.1016/s0074-7696(08)62759-x] [Citation(s) in RCA: 61] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
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Brzin J, Kopitar M, Turk V, Machleidt W. Protein inhibitors of cysteine proteinases. I. Isolation and characterization of stefin, a cytosolic protein inhibitor of cysteine proteinases from human polymorphonuclear granulocytes. HOPPE-SEYLER'S ZEITSCHRIFT FUR PHYSIOLOGISCHE CHEMIE 1983; 364:1475-80. [PMID: 6689311 DOI: 10.1515/bchm2.1983.364.2.1475] [Citation(s) in RCA: 86] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
A protein inhibitor of cysteine proteinases, "stefin", was purified from cytosol of human polymorphonuclear granulocytes. Affinity chromatography on carboxymethylated papain-Sepharose was used as the first step, followed by ion exchange chromatography on DEAE-Sephacel, which resolved four inhibitory peaks. The main peak, comprising approx. 80% of total inhibitory activity was characterized. It was found to be a homogenous protein with an apparent molecular mass slightly lower than that of cytochrome c and with an isoelectric point of 4.65. The inhibitor inhibits papain at a molar ratio of 1:1 as well as cathepsin B and H, but it does not inhibit serine and aspartic proteinases. It is stable at elevated temperature and in alkaline pH, but looses its activity in acid pH. Oxidized glutathione has no effect on its inhibitory activity.
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Rat alpha 1-cysteine proteinase inhibitor. An acute phase reactant identical with alpha 1 acute phase globulin. J Biol Chem 1983. [DOI: 10.1016/s0021-9258(17)44195-0] [Citation(s) in RCA: 64] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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Killackey JJ, Roughley PJ, Mort JS. Proteinase inhibitors of human articular cartilage. COLLAGEN AND RELATED RESEARCH 1983; 3:419-30. [PMID: 6641126 DOI: 10.1016/s0174-173x(83)80022-3] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Extracts of human articular cartilage contain a variety of inhibitors to serine, cysteine and metallo-proteinases. By gel filtration chromatography, inhibitory activity towards serine proteinases was resolved into two components of apparent molecular weights 62,000 and 12,000 daltons; whereas inhibitory activity towards cysteine proteinases eluted with an apparent molecular weight of 13,000 daltons. In both cases the low molecular weight inhibitors were further resolved into two components by ion-exchange chromatography. Inhibitory activity towards metallo-proteinases resolved into two components of apparent molecular weights 35,000 and 25,000 daltons. No inhibitor of aspartic proteinases was detected. Although most of the inhibitory activities to serine and cysteine proteinases could be extracted from cartilage with 1 M NaCl, the complete removal of metalloproteinase inhibitory activities required extraction with 4 M guanidinium chloride. This suggests that they are more strongly associated with the cartilage.
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Hirado M, Niinobe M, Fujii S. Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum. BIOCHIMICA ET BIOPHYSICA ACTA 1983; 757:196-201. [PMID: 6601964 DOI: 10.1016/0304-4165(83)90109-5] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Two kinds of cysteine proteinase inhibitor (Mr 145 000 and Mr 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.
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