Amino acid sequence of bovine spleen cathepsin B (original) (raw)

Purification and some properties of buffalo spleen cathepsin B

Journal of Biosciences, 1989

Purification of cathepsin Β from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffaloenzyme is similar to cathepsin Β from other tissues with respect to these properties.

Catalytic and physico-chemical characteristics of goat spleen cathepsin B

IUBMB Life, 1997

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15, 785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa . Among the various substrates tested, Z-Phe-Arg-MCA with a K of 0.058 mM and hemoglobin with a K of 1.449 ~tM were the most m preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, a_nti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. !mmunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.

Primary structure of bovine cathepsin S Comparison to cathepsins L, H, B and papain

FEBS Letters, 1991

The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (J-CR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3' untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and I3 are 56%, 47% and 31% respectively.

Nucleotide sequence of bovine preprocathepsin B

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993

A cDNA encoding bovine procathepsin B was isolated. The deduced amino acid sequence revealed that a stop (TAG) codon, instead of a Trp-257 codon (TGG), generates in bovine a cathepsin B precursor four amino acids shorter than in other species. Because micro-heterogeneities were previously reported in the cathepsin B primary structure, scquence polymorphism in the protein coding region was then investigated by PCR sequencing of genomic fragments and RNase protection assays. Experiments performed with 12-15 animals of three breeds did not reveal any difference with our cDNA sequence. We conclude that sequence polymorphism in bovine cathepsin B is a rare event, and can only result from the expression of different alleles of a unique gene. Lysosomal proteinases are thought to play a major role in protein catabolism, especially the cysteine proteinases cathepsin B (EC 3.4.22.1), H (EC 3.4.22.16) and L (EC 3.4.22.15) which have been extensively characterized at structural and functional levels [1]. Regarding cathepsin B, purification procedures and amino acid sequences were described for rat [2], human [3] and bovine [4] species. Recent reports, describing a three-dimensional model [5] and the X-ray crystal structure [6] of mature cathepsin B, represent important advances in understanding cysteine proteinase enzymatic properties. Characterization of cDNAs encoding human [7], mouse [7,8] and rat cathepsin B [9] has revealed that this cysteine proteinase is synthesized as a preproenzyme which undergoes a post-translational processing during its transport to the lysosomal compartment (reviewed in Ref. 10). Under physiological conditions, a tissue-specific distribution of cathepsins has been observed [11,12], but the levels of the proteins are not always related to those of mRNAs [13]. Between species, variations in cathepsin activities have also been pointed out [14].

Purification and amino acid sequence of chicken liver cathepsin L

Biochemistry, 1987

Biochemistry 1987, 26, 5689-5695 5689 the extent that a comparison of the lysed and intact cell systems is valid, there are several possible explanations for this discrepancy. It is possible that the level of 5-HPETE generated is sufficient to deplete glutathione, the principal reducing agent for peroxides. This is unlikely, however, because the intracellular concentration of glutathione is about 5 mM (Egan & Gale, 1985) and the total intracellular concentration of 5lipoxygenase products from endogenous arachidonic acid reaches only about 0.08 mM (Sun & McGuire, 1984; calculated assuming 5 X lo8 cells/g wet weight). It is possible that the 5-HPETE that is formed in intact cells is segregated from the peroxidases so that the rapid rate of reduction of 5-HPETE in vitro is not indicative of the in vivo rate. It is also possible that the intrinsic partitioning of 5-HPETE on the enzyme between dissociation and conversion to LTA, is different for 5-lipoxygenase in its native intracellular state and the relative inefficiency observed in vitro is an artifact. We cannot at present provide evidence for either of these possibilities.

Amino acid sequence of human liver cathepsin B

FEBS Letters, 1985

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.

Isolation and characterisation of cathepsin-B from bovine pancreas

Journal of Biosciences, 1979

Α simple procedure for the isolation of cathepsin B from bovine pan creas employing ammonium sulphate fractionation, DEAE cellulose chromatography and Sephadex G 200 gel filtration is described. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight as determined by gel filtration of the enzyme was 26,850. Its K m and V max values were 12·8 mM and 0·303 µmol/min/mg, respectively. The K i for iodoacetamide was 0·16 mM.

On the tissue/species dependence of cathepsin B isozymes

Molecular and cellular biochemistry, 1997

Characterization of cathepsin B from buffalo kidney and goat spleen showed the presence of isozymes in case of the goat spleen (GSCB-I and GSCB-II) whereas cathepsin B from buffalo kidney exhibited only one form (BKCB). The molecular weights determined by SDS-PAGE for GSCB-I, GSCB-II, and BKCB were 25.7, 26.6 and 25.5 kDa respectively. The kinetic parameters (Km and Vmax) of GSCB-I showed close similarities with BKCB against alpha-N-benzoyl-DL-arginine-2-napthylamide whereas GSCB-II was closer to the buffalo enzyme with regards to its activity against Z-Arg-Arg-MCA and Z-Phe-Arg-MCA. All the three enzymes had similar sensitivities towards urea, antipain and leupeptin. However, clear differences were observed in the inhibition patterns of the enzyme with iodoacetic acid and iodoacetamide. Differences in the kinetic, immunogenic and some catalytic properties of GSCB-I and II, which had similarities with regard to most of their physico-chemical properties, were considered to be due to ...

Amino acid sequences of the human kidney cathepsins H and L

FEBS Letters, 1988

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an M, of 25 116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an IV, of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.