Cathepsin D. Purification of isoenzymes from human and chicken liver.
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ABSTRACT:
SUBMITTER: Barrett AJ
PROVIDER: S-EPMC1178965 | biostudies-other | 1970 Apr
REPOSITORIES: biostudies-other
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Project description:Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.
| S-EPMC1144697 | biostudies-other
Project description:Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.
| S-EPMC1132806 | biostudies-other
Project description:The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size.A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.
| S-EPMC2654340 | biostudies-literature
Project description:Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.
| S-EPMC1153198 | biostudies-other
Project description:1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.
| S-EPMC1172807 | biostudies-other
Project description:The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.
| S-EPMC3989067 | biostudies-literature
Project description:Isoelectric focusing of a cytosol fraction from human foetal liver revealed the existence of an acidic and a basic isoenzyme of GSH transferase. The acidic and basic forms of GSH transferase were purified in good yield by use of ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on S-hexyl-GSH coupled to epoxy-activated Sepharose 6B. The content of the acidic and the basic isoenzymes of GSH transferase together was calculated to constitute 1-2% of the soluble proteins in the hepatic cytoplasm. Physical, catalytic and immunological analyses of the acidic and the basic isoenzymes from foetal liver demonstrated unambiguously that the two forms are different structures with distinct properties. On the other hand, the results show clearly extensive similarities between the foetal acidic transferase and transferase pi from human placenta as well as between the foetal basic form and the basic isoenzymes isolated from adult liver. An exception is that both foetal enzymes seem to be considerably more efficient in catalysing the conjugation of GSH with styrene 7,8-epoxide than the corresponding adult forms of GSH transferase.
| S-EPMC1146750 | biostudies-other
Project description:Aspergillus fumigatus sterol 14-? demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K(s), 8.6 ?M) and eburicol (K(s), 22.6 ?M). Membrane suspensions of AF51A bound to both lanosterol (K(s), 3.1 ?M) and eburicol (K(s), 4.1 ?M). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K(d) (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 ?M, respectively, in comparison with a K(d) value of 4 ?M for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K(d) values ranging from 1 ?M for itraconazole to 11.9 ?M for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.
| S-EPMC2944604 | biostudies-literature
Project description:1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of alpha-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.
| S-EPMC1177541 | biostudies-other
Project description:1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.
| S-EPMC1165961 | biostudies-other