Rat Liver Cathepsin Research Articles

Unique biochemical and biological features of cathepsin D in rodent lymphoid tissues.

Cathepsin D, an enzyme consistently found to be lysosomal in many cells, has an unusual localization in rat thoracic duct lymphocytes (TDL). After fractionation of homogenates of rat TDL, most of the enzyme activity, as measured at pH 3.6 on denatured bovine hemoglobin, is distributed differently from the other lysosomal enzymes. The enzyme also has some unique properties: it is not inhibited by an antiserum inhibitory for rat liver cathepsin D; it exists in two molecular weight forms (approximately 45,000 and approximately 95,000) both of which have a higher specific activity than rat liver cathepsin D, as determined by studies using the irreversible inhibitor, sodium pepstatin; the high molecular weight form converts to the low molecular weight form after treatment with beta-mercaptoethanol without any loss in activity. These enzymes appear to be restricted to rodent lymphoid tissues. Reasons for considering them to be a type of cathepsin D are given in the text.

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.

SOME PROPERTIES OF CATHEPSINS CHEMICALLY FIXED TO CARRIERS

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.

Characterization of rat liver lysosomal cathepsin A1

1. 1. Highly purified rat liver cathepsin A1 was shown to be a carboxypeptidase with a high degree of specificity for aromatic and hydrophobic amino acids. 2. 2. In lysosomal soluble fractions, cathepsin A1 was partially resposible for the hydrolysis of the model substrates, N- benzyloxycarbonyl (Cbz)-Gly-Phe, Cbz-Glu-Phe, and Ac-Phe-Tyr. Dipeptides and tripeptides were not hydrolyzed, but N-blocked dipeptides that contain an aromatic or hydrophobic amino acid were hydrolyzed. 3. 3. The optimum hydrolysis of N-blocked dipeptides by cathepsin A1 was at pH 5.0–5.8, with K m values in the range of 1.2–54 mM. 4. 4. The hexapeptide Leu-Trp-Met-Arg-Phe-Ala, glucagon, and insulin B chain were partially hydrolyzed by cathepsin A1 with the release of amino acids from the C-terminal end. These peptides all contain hydrophobic portions near the C-terminal end. Less hydrophobic peptides, such as insulin A chain and His-Ser-Gln-Gly-Thr-Phe, were not hydrolyzed. Very little hydrolysis of denatured hemoglobin was found. 5. 5. Cathepsin A1 was activated by halide ions and inhibited by Ag + and Hg 2+.

Effect of Cathepsins D from Normal and Malignant Tissues on Synthetic Peptides

Abstract The activities of cathepsins D from rat liver and from transplantable rat sarcoma tissue on synthetic peptides were studied. It was shown that, although the two cathepsins are similar in some properties, their binding sites have different structures. Sarcoma cathepsin can split peptides that contain either aromatic or dicarboxylic amino acid residues. Substrates for this enzyme must have peptide chains of at least 5 amino acid residues. Sarcoma cathepsin D displays highest affinity for those substrates that contain dicarboxylic amino acid residues on both sides of the peptide bond cleaved. Replacement of 1 or 2 dicarboxylic amino acid residues by a phenylalanyl residue decreased the apparent affinity of the enzyme for the substrate. Of several structures tested as substrates, the cathepsin D from rat liver has been observed to split only those peptides that contain an aromatic amino acid residue. Both enzymes studied can, like aminopeptidase, split off the NH2-terminal alanyl residue from the peptide Ala-Phe-Gly-Leu-Asp-Val. The synthetic substrate for pepsin, acetyl-Phe-Tyr, inhibits competitively rat liver cathepsin but has no effect on sarcoma cathepsin.

Properties of cathepsin C from rat liver

1. 1. Rat liver cathepsin C (EC 3.4.4.9) was purified 1790-fold over the homogenate activity by a purification scheme that combined the techniques of centrifugation, acid precipitation, (NH 4) 2SO 4 precipitation, and Sephadex G-200, DEAE-cellulose, and CM-cellulose column fractionations. 2. 2. The specific activity of the most purified CM-fraction with Gly-Tyr-NH 2 as substrate was 91.6 μmoles of hydroxamate produced per mg of protein per min, which is higher than previously reported values. 3. 3. The K m for Gly-Tyr-NH 2 was 6 mM when determined by hydroxamate formation, and that for Val-Leu-NH 2 was 2.5 mM. 4. 4. The requirements for −SH compounds and for halide ions for activity were absolute. A dithioerythritol concentration of 25 mM and a Cl − or Br − concentration of 10 mM were required for full activation. 5. 5. The enzyme catalyzed a hydrolysis reaction at pH 5, but the polymerization reaction catalyzed at pH 7–8 occurred at a greater rate. 6. 6. The substrate specificity of cathepsin C was broader than that reported in older literature and the enzyme was confirmed to be a dipeptidyl aminopeptidase that acts upon a number of dipeptide amides and tripeptides. The most favorable substrates were tripeptides or dipeptide amides that have as the NH 2-terminal group a small residue and that have the aliphatic residue leucine at the penultimate position.

Relationship of rat liver cathepsins to autolytic degradation of proteins of the liver cell nucleus during isolation: Intracellular distribution and properties of the cathepsins

The distribution and some properties of the liver cell cathepsins have been studied. Cathepsins A, B and C as well as another type of protease were demonstrated in nuclei and the mitochondrial fraction of liver cells, and saline and HCl extracts of all types of aqueous as well as non-aqueous preparations of nuclei that were investigated showed high degrees of proteolytic activity. This makes the characterization of protein fractions isolated from the nuclei somewhat hazardous, owing to the difficulty of avoiding enzymatic degradation during fractionation procedures. NaCl and HCl extracts from Behrens' type nuclei showed even higher proteolytic activity than corresponding extracts of nuclei isolated at pH 5.8 in aqueous media. This finding constitutes some evidence for the natural occurrence of cathepsins within the nucleus of the living cell.