Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

Legumain from bovine kidney: its purification, molecular cloning, immunohistochemical localization and degradation of annexin II and vitamin D-binding protein

Legumain (asparaginyl endopeptidase) was purified to homogeneity from bovine kidneys. The molecular mass of the purified enzyme was calculated to be 34000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol. The enzyme rapidly hydrolyzed the substrate Z-Ala-Ala-Asn-MCA and was strongly inhibited by N-ethylmaleimide, p-chloromercuribenzene-sulfonic acid, Hg(2+) and Cu(2+). The amino acid sequence of the first 26 residues of the enzyme was Gly-Gly-Lys-His-Trp-Val-Val-Ile-Val-Ala-Gly-Ser-Asn-Gly-Gln-Tyr-Asn-Tyr-Arg-His-Gln-Ala-Phe-Ala-Asp-His-. This sequence is highly homologous to the sequences in the N-terminal of pig kidney legumain. We screened a bovine kidney cortex cDNA library using a DNA probe that originated from rat legumain, and we determined the bovine kidney cDNA structure and deduced the amino acid sequence. The cDNA is composed 1934 bp and encodes 433 amino acids in the coding region. The enzyme was strongly stained in the proximal tubules of the rat kidney in an immunohistochemical study. Vitamin D-binding protein which is known to be a ligand to megalin existing in the proximal tubules, was cleaved in a limited proteolytic manner by bovine kidney legumain. These results suggested that legumain contributes to the processing of macromolecules absorbed by proximal tubule cells. The enzyme also cleaved an N-terminal synthetic peptide of bovine annexin II (Gly(24)-Ser-Val-Lys-Ala-Tyr-Thr(30)-Asn-Phe-Asp-Ala-Glu(35)-Arg-Asp(37)) at a position between Asn(31) and Phe(32). The amino-terminal domain of annexin II has p11 subunit binding sites and phosphorylation sites for both pp60(src) and protein kinase C. This suggests that legumain plays an important role in inactivation and degradation of annexin II, which is abundant in the receptor-recycling compartments of endosomes/lysosomes.     

Calcium-dependent affinity purification of transglutaminase from rat liver cytosol

Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

Dipeptidyl peptidase III: a multifaceted oligopeptide N-end cutter

Dipeptidyl peptidase III (DPP III), the sole member and representative of the M49 family of metallopeptidases, is a zinc-dependent aminopeptidase. It sequentially hydrolyses dipeptides from the N-terminal of oligopeptides ranging from three to 10 amino acid residues. Although implicated in an array of pathophysiological phenomena, the precise function of this peptidase is still unclear. However, a number of studies advocate its contribution in terminal stages of protein turnover. Altered expression of DPP III which suggests its involvement in primary ovarian carcinoma, oxidative stress (Nrf2 nuclear localization), pain, inflammation and cataractogenesis has recently led to resurgence of interest in delineating the role of the peptidase in these pathophysiological processes. This review article intends to bring forth the latest updates in this arena which may serve as a base for future studies on the peptidase.     

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen > skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.     

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.     

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.     

Partial purification and characterization of a triacyglycerol lipase from rat liver cytosol.

A triacyglycerol lipase (EC 3.1.1.3) was purifiec about 60-fold from rat liver cytosol by delipidation with acetone and ethyl ether, hydroxyapatitie and Sephadex G-100 column chromatographies and isoelectrofocusing electrophoresis. The partially purified enzyme had a molecular weight of approximately 42 000 and an isolectric point of 7.2. The Km for trioleylglycerol was 0.33 mM and the pH optimum was around 8.0. The activity of the enzyme was not dependent on serum lipoproteins, but was stimulated about 2-fold by several proteins such as serum albumin, lipoproteins, gamma-globulin and ovalbumin. The lipase hydrolyzed trioleyglycerol to oleic acid and glycerol. NaCl had no effect on the enzymatic activity. Some physical and kinetic properties of the partially purified lipid-free lipase were different from those of crude non-delipidated lipase and also from those of a neutral triacylglycerol lipase which was recently purified partially from pig liver cytosol (Ledford, J.H. and Alaupovic, P. (1975) Biochim. Biophys. Acta 398, 132-148).     

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells.On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger ‘immuno-staining’ was exhibited in the peripheries of the hepatic lobules than in the central zone.     

Nonserine esterases from rat liver cytosol

An effort to identify the major general esterases of rat liver cytosol that are insensitive to the serine esterase inhibitor paraoxon (diethyl 4-nitrophenyl phosphate) has led to the isolation of a dozen enzymes. Four of these are electrophoretically homogeneous. Although purified on the basis of their hydrolytic activity toward 4-nitrophenyl acetate, each of the enzymes has a very broad and overlapping substrate specificity for aromatic esters. Thiol esters serve as substrates but, within the limits of the methods used, amides are not hydrolyzed.     

Studies on some molecular properties of cytosol 5'-nucleotidase from rat liver

A cytosol 5'-nucleotidase was purified from rat liver. It appeared to be homogeneous on the criteria of polyacrylamide gel electrophoresis. We estimated the approximate molecular weight of the enzyme to be 200 000 and concluded that the enzyme most likely exists in the native state as a tetramer. Our results suggest that adenine nucleotides, which are activators of the enzyme, induce its conformational change.