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Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.

The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)

An antidiuretic hormone-inactivating peptidase located in renal plasma membranes of porcine kidney medulla has been studied. Treatment of antidiuretic hormone (lysine vasopressin) with renal plasma membranes resulted in a progressive loss of biological activity as measured by the rat pressor assay. The reaction of 2,4,6-trinitrobenzenesulfonic acid with released amino groups was employed to follow the peptidase-catalyzed hydrolysis of the hormone. An 83-fold purification of the membrane-bound peptidase was achieved by Lubrol PX solubilization of the membranes followed by DEAE-cellulose, hydroxylopatite, and 8% agarose column chromatography. The molecular weight of the peptidase was 442 000 as determined by 8% agarose gel filtration. An analysis of the antidiuretic hormone hydrolysis products by thin-layer chromatography revealed the presence of trinitrophenyl-glycinamide. The release of glycinamide from the hormone as a function of time was demonstrated. Mg2+ had a slight inhibitory effect and Ca2+ had a strong inhibitory effect on the peptidase activity.

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.