Purification of three aminopeptidases from human maternal serum

Three aminopeptidases (I--III) were purified from maternal serum using sequential chromatographic fractionations. Aminopeptidase I was specific for N-terminal alpha-L-dicarboxylic acid residues and activated by alkaline earth metals (Ba2+, Ca2+, Sr2+). It is concluded that aminopeptidase I is aminopeptidase A (L-alpha-aspartyl-(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7). Aminopeptidase II hydrolysed all tested substrates including L-cystine and Bz-L-cysteine derivatives but preferred L-leucine derivatives. The properties of aminopeptidase II are equal to those described for the cystine aminopeptidase (oxytocinase) (EC 3.4.11.3.). Aminopeptidase III preferred L-alanine derivatives as substrates. It was activated by Co2+, but strongly inhibited by amastatin, puromycin and L-methionine. The characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-).     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Transformation of steroids by fungal spores

Summary The steroid-hydroxylating activity during spore swelling prior to germination (I), during germ tube formation (II) and during branching of the growth hyphae (III) is two to five times that of the mycelium (IV) and control spores. The increased steroid-transforming activity is not correlated with the overall degradation of free amino acid pools but only with that of alanine, glutamate, arginine and proline. A higher ratio NADPH:(NADP⁺+NADPH) was observed in development phases I, II, and III, which indicates a possible role of alanine, glutamate and other amino acids decomposed via glutamate, in NADPH generation and steroid-hydroxylase activation.     

Control of enzyme synthesis in the arginine deiminase pathway of Streptococcus faecalis

The formation of the arginine deiminase pathway enzymes in Streptococcus faecalis ATCC 11700 was investigated. The addition of arginine to growing cells resulted in the coinduction of arginine diminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3). Growth on glucose-arginine or on glucose-fumarate-arginine produced a decrease in the specific activity of the arginine fermentation system. Aeration had a weak repressing effect on the arginine deiminase pathway enzymes in cells growing on arginine as the only added substrate. By contrast, depending on the growth phase, a marked repression of the pathway by oxygen was observed in cells growing on glucose-arginine. We hypothesize that, in S. faecalis, the ATP pool is an important signal in the regulation of the arginine deiminase pathway. Mutants unable to utilize arginine as an energy source, isolated from the wild type, exhibited four distinct phenotypes. In group I the three enzymes of the arginine deiminase pathway were present and probably affected in the arginine uptake system. Group II mutants had no detectable arginine deiminase, whereas group III mutants had low levels of ornithine carbamoyltransferase. Group IV mutants were defective for all three enzymes of the pathway.     

Coordinate repression of arginine aminopeptidase and three enzymes of the arginine deiminase pathway in Streptococcus mitis

Streptococcus mitis contains two arginine aminopeptidases (I and II) as an arginine-supplying system and the arginine deiminase pathway as an arginine-utilizing system. The levels of arginine aminopeptidase I and three enzymes of the arginine deiminase pathway were suppressed by glucose in an apparently coordinate manner. Enzyme II appeared to be constitutive.     

The purification,specificity, and role of dipeptidyl peptidase III inDictyostelium discoideum

Dipeptidyl peptidase III was purified from culminating cells ofDictyostelium discoideum to apparent homogeneity by salt fractionation, isoelectric focusing, hydrophobic interaction, gel filtration, and ion-exchange chromatography. The enzyme was also found in and purified from cells growing on bacteria or in axenic media. The enzyme from the growth phase was indistinguishable from the culmination enzyme during all purification procedures. Thus dipeptidyl peptidase III is not a developmentally associated enzyme and probably is involved in peptide breakdown throughout the life cycle. The enzyme cleaved arginyl-arginyl-2-naphthylamide most effectively and showed no action on leucyl-glycyl-naphthylamide, thus resembling the mammalian enzyme and differing from the yeast enzyme. Aspartyl-arginyl-naphthylamide was cleaved at 6% of the rate observed with arginyl-arginyl-naphthylamide and the enzymein vivo might therefore be capable of removing the N-terminal peptide (aspartyl-arginine) from peptides such as angiotensins I and II. It showed no aminopeptidase or endopeptidase activity. Angiotensin III was an effective inhibitor of the enzyme; peptides with the N-terminal sequences Gly-Gly, Gly-Arg, Arg-Val, and Gly-His inhibited the enzyme weakly.     

Aminopeptidases of Bacillus subtilis.

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.     

Conversion of brain angiotensin II to angiotensin III is critical for pressor response in rats

The present investigation measured the relative pressor potencies of intracerebroventricularly infused ANG II, ANG III, and the metabolically resistant analogs d-Asp(1)ANG II and d-Arg(1)ANG III in alert freely moving rats. The stability of these analogs was further facilitated by pretreatment with the specific aminopeptidase A inhibitor EC33 or the aminopeptidase N inhibitor PC18. The results indicate that the maximum elevations in mean arterial pressure (MAP) were very similar for each of these compounds across the dose range 1, 10, and 100 pmol/min during a 5-min infusion period. However, d-Asp(1)ANG II revealed significantly extended durations of pressor effects before return to base level MAP. Pretreatment intracerebroventricular infusion with EC33 blocked the pressor activity induced by the subsequent infusion of d-Asp(1)ANG II, whereas EC33 had no effect on the pressor response to subsequent infusion of d-Arg(1)ANG III. In contrast, pretreatment infusion with PC18 extended the duration of the d-Asp(1)ANG II pressor effect by about two to three times and the duration of d-Arg(1)ANG III's effect by approximately 10 to 15 times. Pretreatment with the specific AT(1) receptor antagonist losartan blocked the pressor responses induced by the subsequent infusion of both analogs indicating that they act via the AT(1) receptor subtype. These results suggest that the brain AT(1) receptor may be designed to preferentially respond to ANG III, and ANG III's importance as a centrally active ligand has been underestimated.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Subcellular localization and levels of aminopeptidases and dipeptidase in Saccharomyces cerevisiae.

Three aminopeptidases (L-aminoacyl L-peptide hydrolases, EC 3.4.11) and a single dipeptidase (L-aminoacyl L-amino acid hydrolase, EC 3.4.13) are present in homogenates of Saccharomyces cerevisiae. Bassed on differences in substrate specificity and the sensitivity to Zn2+ activation, methods were developed that allow the selective assay of these enzymes in crude cell extracts. Experiments with isolated vacuoles showed that aminopeptidase I is the only yeast peptidase located in the vacuolar compartment. Aminopeptidase II (the other major aminopeptidase of yeast) seems to be an external enzyme, located mainly outside the plasmalemma. The synthesis of aminopeptidase I is repressed in media containing more than 1% glucose. In the presence of ammonia as the sole nitrogen source its activity is enhanced 3--10-fold when compared to that in cells grown on peptone. In contrast, the levels of aminopeptidase II and dipeptidase are less markedly dependent on growth medium composition. It is concluded that aminopeptidase II facilitates amino acid uptake by degrading peptides extracellularly, whereas aminopeptidase I is involved in intracellular protein degradation.     

Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells

Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus [1-(14)C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.     

Membrane Bound Aminopeptidase B of a Potential Probiotic Pediococcus acidilactici NCDC 252: Purification,Physicochemical and Kinetic Characterization

International Journal of Peptide Research and Therapeutics - An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogeneity from membrane extract of a...     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

Sensitivity in vitro of mature and immature mouse thymocytes to dexamethasone cytotoxicity and its correlation to poly ADP-ribosylation.

Mouse thymocytes were fractionated into heavy (subtype I, 79% of total cell number), medium (subtype II, 18%) and light (subtype III, 3%) ones by Percoll density centrifugation and they were identified as immature (subtype I and II) and mature (subtype III) thymocytes based on their proliferative response to mitogens. Whereas the nuclear activity of poly (ADP-ribose) polymerase (EC 2.4.2.30) in the subtype III was only one half that of denser subtypes, it increased two-fold upon mitogen stimulation. The sensitivity of three thymocyte subtypes to the dexamethasone cytotoxicity, as judged by the extent of the DNA cleavage, depletion of NAD and cell viability, was highest in the subtype I and lowest in the subtype III. The possible involvement of poly ADP-ribosylation in the apoptotic (programmed) cell death during intrathymic development of immature to mature thymocytes is discussed.     

Chloride-insensitive, glycine-phenylalanine-naphthylamide hydrolysis at neutral pH in human skin fibroblasts

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine--phenylalanine--beta-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid--naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependent.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Aminopeptidase A is angiotensinase A. II. Biochemical studies on aminopeptidase A and M in rat kidney homogenate

Biochemical fluorometric methods were used to investigate aminopeptidase A (APA; E.C.3.4.11.7) in the rat kidney homogenate and glomeruli and to compare it with aminopeptidase M (APM; E.C.3.4.11.2). It is shown that APA is a calcium-ion-dependent enzyme, while APM is not. To clarify the functional importance of APA and APM in the kidney, their activities were measured under the influence of angiotensins. Fluorimetric measurements in renal homogenate (with 2-naphthylamide derivatives as substrates), which represents mixed-enzyme tissue preparations containing a variety of peptidases besides APA and APM, showed a Km of 0.13 mM for APA and competitive inhibition of ANG II (K1 = 0.015 mM), and a Km of 0.24 for APM and competitive inhibition by ANG III (K1 = 0.003 mM). The remaining two angiotensins showed non-competitive inhibition of APA (ANG I, III) and APM (ANG I, II) in this preparation. For comparison purposes, fluorometric measurements were performed in microdissected glomeruli which contain only APA. A Km of 0.23 mM for the APA and a competitive inhibition of APA by ANG I and II were determined. Thus it was possible to show biochemically that APA is equivalent to angiotensinase A and that both APA and APM participate in angiotensin degradation in the kidney. APA initiating the breakdown of ANG I and II, and APM possibly continuing it in sequential fashion.     

Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues. Evidence for a new mode of binding to aminopeptidases

The synthesis and inhibition kinetics of a new, potent inhibitor of arginine aminopeptidase (aminopeptidase B; EC 3.4.11.6) are reported. The inhibitor is a reduced isostere of bestatin in which the amide carbonyl is replaced by the methylene (-CH2-) moiety. Analysis of the inhibition of arginine aminopeptidase by this inhibitor according to the method of Lineweaver and Burk yields an unusual noncompetitive double-reciprocal plot. The replot of the slopes versus [inhibitor] is linear (Kis = 66 nM), but the replot of the y intercepts (1/V) versus [inhibitor] is hyperbolic (Kii = 10 nM, Kid = 17 nM). These results provide evidence for a kinetic mechanism in which the inhibitor binds to the S1' and S2' subsites on the enzyme, not the S1 and S1' subsites occupied by dipeptide substrates. Furthermore, structure-activity data for a series of ketomethylene dipeptide isosteres in which the amide (-CONH-) of a dipeptide is replaced with the ketomethylene (-COCH2-) moiety show that the S1 and S1' subsites preferentially bind basic and aromatic side chains, respectively. These results are in agreement with the known substrate specificity of arginine aminopeptidase. The structure-activity data for several bestatin analogues, however, show that these compounds do not bind to the S1 and S1' sites of arginine aminopeptidase. A comparison of the data provides evidence that bestatin inhibits arginine aminopeptidase and possibly other aminopeptidases by binding to the S1' and S2' sites of the enzyme.     

Three Forms of α-Glucosidase from Suspension-cultured Rice Cells

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the three enzymes were inhibited by 11.25 ng of castanospermine. The inhibition was competitive.     

Source of oestrogen in early pregnancy in the mare

Oestrogen secretion was determined by oestrogen conjugate (EC) analysis of urine in three groups of pregnant mares: Group I (N = 6), animals ovariectomized on Day 18-19 of gestation with pregnancy maintained by daily administration of an oral progestagen, altrenogest; Group II (N = 9), untreated, pregnant mares; Group III (N = 5) intact, pregnant mares treated daily with altrenogest. The mean EC concentrations in the ovariectomized mares in Group I increased in a constant linear manner from 17 ng/mg Cr on Day 20 to 291 ng/mg Cr on Day 70, with no apparent surge in oestrogen secretion around Day 39. Mean EC concentrations on Days 33, 39 and 44 were respectively 41, 48, and 73 ng/mg Cr. In the intact mares in Groups II and III (shown in parentheses), the mean urinary EC concentrations were 201 (171) ng/mg Cr between Days 20 and 33 of gestation, increased rapidly from 172 (77) ng/mg Cr on Day 33 to a peak of 1066 (895) ng/mg Cr on Day 39, followed by a decline to 637 (719) ng/mg Cr on Day 44. After Day 44, EC concentrations continued to increase in a linear manner to 1191 (842) ng/mg Cr on Day 70. The mean EC concentrations between Days 20 and 70 in Group I were significantly (P less than 0.05) lower than in mares in Groups II and III. EC concentrations in Group III mares were significantly lower (P less than 0.05) than in Group II mares between Days 28 and 34.(ABSTRACT TRUNCATED AT 250 WORDS)