Dipeptidyl peptidases in the soleus muscle of the rat before and after treatment with 5-hydroxytryptamine

Summary A moderate peptidase activity against l-lysyl-l-proline-4-methoxy--napththylamide was detected histochemically in unfixed sections of soleus muscle fibres of inbred male Wistar rats using two variants of the semipermeable membrane technique. One variant involved simultaneous coupling with tetrazotised 3,3-dimethoxybenzidine, the other post-coupling. The activity at pH 6 increased approximately three-fold in many fibres showing signs of insult in rats that had been given a single low dose of 5-hydroxytryptamine (10 mg/kg body weight) 48–72 h earlier. The hydroxytryptamine treatment was found to induce a selective myopathy. Some of the increased peptidase activity within insulted muscle fibres appeared to arise from invading mononuclear cells, but the majority seemed endogenous to muscle fibres. The peptidase activity persisted in some fibres 21–28 days after 5-hydroxytryptamine administration, by which time the whole muscle appeared histologically normal.The variation of the activity of the peptidase with pH in the presence of various inhibitors was investigated in both control and insulted muscle fibres. From its sensitivity and behaviour towards Zn²⁺, Hg²⁺, Cu²⁺, puromycin, benzethonium chloride and phenylmethylsulphonyl fluoride and its indifference towards Co²⁺, Cd²⁺; Mn²⁺ and o-phenanthroline, it is concluded that the activity can be attributed to a mixture of at least two peptidases, dipeptidyl peptidase II and an unidentified neutral dipeptidyl peptidase. The possible role of the peptidase(s) in muscle regeneration in discussed.Dedicated to Professor Dr. T.H. Schicbler on the occasion of his 65th birthday     

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.     

Peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses derived from Shumiya cataract rats

The role of proteolytic enzymes in Shumiya cataract rats in alterations to lens proteins during cataract formation was studied immunohistochemically using antibodies against exopeptidases, such as lysosomal dipeptidyl peptidase II (DPP II), cytosolic dipeptidyl peptidase III, and soluble and membrane-bound alanyl aminopeptidases, and against cytosolic endopeptidases such as mu- and m-calpains, and 20S proteasome. AlphaB-crystallin was detected as a proteolytic marker in the lenses. A constant immunoreactivity against all the antibodies employed was observed in the lens epithelium independent of the strain and age of the rats. A weak immunoreactivity against exo- and endopeptidases and an intense reactivity against alphaB-crystallin were observed in the lens fibres of control rats at all ages. The immunoreactivity of these peptidases in lens fibres increased with age in cataract rats, but that of alphaB-crystallin decreased. No reactivity against exo- and endopeptidases was seen in the perinuclear region of lenses of control rats at all ages or in Shumiya cataract rats at 8 and 10 weeks of age, but an intense reactivity against these peptidases was observed in the lens perinuclear region of lenses in cataract rats at 12 and 14 weeks of age. AlphaB-crystallin immunoreactivity was observed with ordered striations in the lens perinuclear region of all control rats whereas the striations in this area of cataract rat lens were disorganized. Membrane-bound alanyl aminopeptidase was detected feebly in the lens epithelium and fibres of both types of rat at all weeks of age. These findings indicate that exo- and endopeptidases, except for membrane-bound alanyl aminopeptidase, are expressed intensively and are age-dependent. Conversely, the amount of alphaB-crystallin decreased with age in lens fibres of cataract rats. Calpains (mu- and m-), 20S proteasome, dipeptidyl peptidases II and III and soluble alanyl aminopeptidase are thought to induce lens opacification kinetically during cataract formation in Shumiya cataract rats through the intracellular turnover of lens proteins.     

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.     

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.     

Separation of rat muscle aminopeptidases.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Partial purification and characterization of the oxytocin-inactivating enzymes from chicken liver.

1. Two enzymes acting on the linear portion of oxytocin: carboxamidopeptidase (releasing Gly . NH2) and prolyl peptidase (releasing Leu-Gly . NH2) were identified in the cytoplasmic fraction of chicken liver. 2. Carboxamidopeptidase was purified 134-fold with a 23% yield, and prolyl peptiase 71-fold with a 20% yield. The specific activity of the final preparations was 181 and 96 microU/mg protein, respectively. 3. The optimum pH for carboxamidopeptidase was 6.0--6.5 and for prolyl peptidase, 7.5. Carboxamidopeptidase activity was inhibited by Mn2+, Zn2+, Ca2+, Co2+, and stimulated by EDTA; the activity of prolyl peptidase was inhibited by Zn2+ and Mn2+. The Km value of both enzymes for oxytocin was 1.5--2.4 microM.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

A dipeptidyl carboxypeptidase in brain synaptic membranes active in the metabolism of enkephalin containing peptides

A dipeptidyl carboxypeptidase activity has been localized in synaptic plasma membranes which have been prepared from isolated rat brain cortical synaptosomes. The specificity of this proteolytic activity towards various synthetic and biological active peptides is compared to the peptidase activities of intact synaptosomes. In contrast to the synaptosomal peptidases which are capable of cleaving all peptide bonds of Met-enkephalin-Arg6-Phe7 the peptidase activity associated with the synaptic plasma membrane exclusively hydrolyses a dipeptide from the carboxyl terminus of all hepta- and hexapeptides tested. The fact that this dipeptidyl carboxypeptidase does not cleave the Gly3-Phe4 peptide bond of Met-enkephalin suggests that this enzyme is different from "enkephalinase". The synaptic membrane dipeptidyl carboxypeptidase is inhibited by metal chelating agents and thiols but is not affected by compounds known to inhibit serine proteases, thermolysin and "enkephalinase".     

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.

We have determined the half-lives and amounts per hepatocyte of the polymeric IgA receptor (pIgA-R) and four rat hepatocyte plasma membrane proteins and subsequently have predicted their rates of synthesis and possible routes of degradation. Using in vivo pulse-chase metabolic labeling with L-[35S]cysteine, we found that the pIgA-R had an apparent half-life of 1.1 h. Additional metabolic labeling experiments showed that CE9, HA4, and HA321 had apparent half-lives of 4-5 days, and dipeptidyl peptidase IV had an apparent half-life of 9 days. To quantify the amount of each protein per hepatocyte, homogenates and a standard curve of purified protein were compared by immunoblotting. We found that these proteins were present at 1-8 x 10(6) molecules/hepatocyte. The calculated rate of synthesis for pIgA-R was 1.6 x 10(6) molecules/hepatocyte/h, whereas the others were synthesized at much lower rates (0.9-5 x 10(4) molecules/hepatocyte/h). Using immunoblot analysis, we found that pIgA-R was released into bile at a rate of 30%/h (700%/day), whereas dipeptidyl peptidase IV and HA4 were released at a rate of 2-3%/day. While the majority of the loss of pIgA-R from hepatocytes occurred by release into the bile, less than 30% of the degradation of dipeptidyl peptidase IV and HA4 could be accounted for by this pathway, suggesting that the remaining molecules must be retrieved from the apical surface before degradation.     

Expression, purification, and kinetic characterization of full-length human fibroblast activation protein

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.     

Apical plasma membrane-bound enzymes of rabbit uterine epithelium. Pattern changes during the periimplantation phase

In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5-8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals. All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the "receptive state", 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.