Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation

Popović, Tatjana; Puizdar, Vida; Ritonja, Anka; Brzin, Jože

doi:10.1016/0378-4347(95)00555-2

Cited by 17 publications

(15 citation statements)

References 50 publications

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“…␤-Trypsin (EC 3.4.21.4) was prepared from type IX trypsin (Sigma) as described (19). Cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16), and cathepsin L (EC 3.4.22.15) were purified from human kidney by the method already described (20 Inhibitor Purification-Frozen fruit bodies of C. nebularis (500 g, fresh weight) were homogenized in 1000 ml of Tris/HCl buffer, pH 7.5, containing 0.5 M NaCl (Buffer A). The homogenate was centrifuged at 8000 ϫ g for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

Brzin¹,

Rogelj²,

Popović³

et al. 2000

Journal of Biological Chemistry

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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. Cysteine proteinases are involved in a diverse array of functions involving specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals. Their activity is regulated by limited proteolysis of inactive precursors (1, 2), by pH and redox potential of the surroundings, and tight binding with proteinaceous inhibitors (3).Five structurally different groups of protein cysteine proteinase inhibitors have been reported: cystatins (4), thyroglobulin type-1 domain inhibitors or thyropins (5), soybean trypsin inhibitor-like inhibitors of cysteine proteinases from potato (6), pineapple inhibitors of cysteine proteinases (7,8), and very recently, inhibitors of cysteine proteinases homologous to propeptide regions of cysteine proteinases (9). So far, only the mechanism of interaction of the cystatin superfamily of inhibitors has been elucidated (10, 11), followed recently by that of thyropins (12), but overall there is very little information available on all other cysteine proteinase inhibitors concerning specificity, kinetics, and mode of binding.Since cysteine proteinases in mammals have been implicated also in many pathological events, such as tumor invasion and metastasizing cancer (13), bone resorption (14), periodontitis (15), and rheumatoid arthritis (16), there is a need for new specific, efficient, and accessible inhibitors of the enzymes responsible for diagnosis and treatment of these conditions. Fungi (Mycophyta) have been used for religious, medical, and other purposes since ancient times. To our knowledge, no protein cysteine proteinase inhibitors have been characterized in higher fungi (Basydiomyceta), popularly called mushrooms. We report here the identification, some properties, and cloning of a new proteinaceous cysteine proteinase inhibitor from Clitocybe nebularis fruit bodies, which we have called clitocypin, a member of what is very likely a new structural superfamily of cysteine proteinase inhibitors. EXPERIMENTAL PROCEDURESFungal Material-Edible mushrooms, C. nebularis, were collected in their natural habitat in a forest in November and frozen at Ϫ20 or Ϫ70°C until use. A specimen is deposited at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Ljubljana.Chemicals and Enzymes-Iodoacetate and benzoyl-A...

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Section: Methodsmentioning

confidence: 99%

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

Brzin¹,

Rogelj²,

Popović³

et al. 2000

Journal of Biological Chemistry

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“…Cathepsin containing fraction was isolated as described in [18] with minor modifications. Rat brain supernatant was acidified to pH 4.0, and ammonium sulfate was added up to 20% saturation.…”

Section: Methodsmentioning

confidence: 99%

10.1007/s10541-008-3014-5

2000

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332Abbreviations: AMC) 7 amino 4 methylcoumarin; CA 074) N [L 3 trans (propylcarbamoyl)oxirane 2 carbonyl] L isoleucyl L proline; DTT) dithiothreitol; MALDI TOF MS) matrix assisted laser desorption/ionization with time of flight mass spectrometry. Abstract-Weshow that an enzyme exists in rat brain capable of cleaving the caspase 3 specific peptide substrate Ac DEVD AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI TOF MS. The enzyme possessing "acidic" DEVDase activ ity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase 3 sub strates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase 3 substrates in brain cells.

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“…DPP-I from rabbit, S. japonicum and rat also showed optimal activity at similar pH. pH optima of 6.0 or slightly higher were also noted in R. norvegicu, M. musculus, B. taurus and P. falciparum naeus japonicus showed maximum activity at an acidic pH range (5.0-5.5) [25,27,28,32,35,[38][39][40][41][42]. DPP-I was found to be stable in a narrow pH range of 6-7.5 with >90% activity, and >70% activity was retained at pH 5.0 and 8.0.…”

Section: Ph Optima and Stabilitymentioning

confidence: 97%

“…The molecular weight of plant DPP-I is close to the molecular weight of matured DPP-I from flatworm [22]. However, the molecular weight of mammalian (humans, rabbit and rat) DPP-I was reported to range from 180 to 210 kDa with subunits of 21-24 kDa [25][26][27][28][32][33][34][35]. However, gene expression studies predicted the monomeric nature of DPP-I from humans, rat, mice, Plasmodium falciparum and fluke, which was processed into prodomain, heavy chain and light chain on maturation [29,36].…”

Section: Purification Of Enzymementioning

confidence: 99%

“…Our results are in agreement with previous reports on the temperature optima of DPP-I from rabbit, humans and pig, which ranged between 45 and 55 • C [25,26,28]. However, DPP-I homologues from several other species such as humans (liver and kidney cells), rat (liver, brain, bone), B. taurus (spleen), dog (mast cell), P. falciparum, S. japonicum and M. japonicus had an optimum temperature of 37 • C [30][31][32][38][39][40][41][42][43][44][45]. The activation energy (E a ) of plant DPP-I was calculated by the Arrhenius plot and was found to be 21.763 KJ/mol for Gly-Arg-4m␤NA.…”

Section: Temperature Optima and Stabilitymentioning

confidence: 99%

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Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds

Khaket

Dhanda

Jodha

et al. 2016

Process Biochemistry

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Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation

Cited by 17 publications

References 50 publications

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

10.1007/s10541-008-3014-5

Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds

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