Comparative characteristics of soluble and membrane brain aminopeptidases. II. Substrate specificity

Comparative studies on substrate specificity of the soluble and membrane-bound aminopeptidases from bovine brain were carried out. A series of p-nitroanilides and beta-naphthylamides of amino acids, di- and tripeptides with the aminoterminal phenylalanine residue, as well as a biologically active pentapeptide--[Leu5]enkephalin--were used as substrates. The soluble and membrane-bound aminopeptidases manifested identical specificity towards the employed substrates. The aminopeptidases were equally effective towards the p-nitroanilides of amino acids and peptides, whereas beta-naphthylamides were more susceptible to hydrolysis by both aminopeptidases than p-nitroanilides and peptides. Taking into account physico-chemical characteristics of these enzymes, it was concluded that the soluble and membrane-bound aminopeptidases are quite similar or perhaps identical. Their role in the regulation of nervous system functioning was discussed. A comparison of specificities for brain aminopeptidases and leucine aminopeptidase from bovine lens led to the conclusion that they belong to different groups. This feature allows planning the synthesis of selective inhibitors.     

Comparative distribution of glutamyl and aspartyl aminopeptidase activities in mouse organs.

To evaluate the functional role of glutamyl and aspartyl aminopeptidases, their soluble and membrane-bound activities were measured simultaneously in several tissues of normal mice using arylamide derivatives as substrates. Although the soluble aspartyl aminopeptidase activity showed its highest levels in the testicle, the rest of the activities presented their highest levels in the kidney. Different patterns of distribution were observed for glutamyl and aspartyl aminopeptidase activities and also for soluble and membrane-bound aspartyl aminopeptidase activities. However no major differences were observed between soluble and membrane-bound glutamyl aminopeptidase activities. This unequal distribution suggests that the use of arylamide derivatives as substrates is a sensitive method that distinguishes between these enzymatic activities. The results also suggest different functions for soluble and membrane-bound aspartyl aminopeptidase activities, and for glutamyl and aspartyl aminopeptidase activities.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

Soluble enkephalin-degrading enzymes released by lymphomic and erythroleukaemic cell lines

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of³H-leucine enkephalin in the presence of cell-free supernatants obtained from these lines. Results indicate that leu-enkephalin is rapidly degraded in the presence of these supernatants, and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. To characterize the factors involved in leu-enkephalin degradation, cell supernatants were analyzed by ion exchange and by steric exclusion chromatography. Data obtained indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In all three lines, these enzymes are represented by a considerable number of distinct activities. The sizable number of soluble enzymes identified and the signficant total activity observed suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

A simple one-step procedure for the separation of calpain I, calpain II and calpastatin.

The soluble fraction from rabbit brain was adsorbed on a column of phenyl-Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transported soluble proteins and rapidly transported membrane-bound proteins, as well as casein.     

Native agarose-polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase, and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains

Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippopha? host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Detection and localization of two hydrogenases in Methylococcus capsulatus (Bath) and their potential role in methane metabolism

Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD(+) with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed.     

Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Effects of changes in hydromineral balance on rat brain aspartyl, arginyl, and alanyl aminopeptidase activities.

In order to examine the possible relationship between the processing and inactivation roles of aminopeptidases and the disruption of water-electrolyte balance, we measured the activities of aspartyl aminopeptidase (Asp-Ap), arginyl aminopeptidase (Arg-Ap) and alanyl aminopeptidase (Ala-Ap) in certain brain areas (hypothalamus, hippocampus, thalamus and brain cortices) and in the pituitary gland in several models of hydrosaline change. The activity of hypothalamic membrane-bound Asp-Ap significantly decreased (more than 50%) following treatments which induced a hypovolemic state. Aminopeptidase M activity (membrane-bound Ala-Ap activity with low sensitivity to puromycin) was also significantly decreased by 53 % in the thalamus of rats under conditions of hypovolemia plus hyperosmolality in comparison to the control group. These results indicate that aminopeptidases in the central nervous system may be involved in the physiological regulation of hydromineral balance.     

Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin-Like Metalloendopeptidase (Enkephalinase), Angiotensin-Converting Enzyme, and Other Unidentified Peptidases

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.     

Acid and alkaline phosphatases of Capnocytophaga species. I. Production and cytological localization of the enzymes

The two hydrolytic enzymes, acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase, of the three types species of Capnocytophaga were examined. Both enzymes were produced constitutively, with their activity highest in C. ochracea strain 25. These two degradative enzymes (approximately 10% of the total activity) were released into the growth medium during the latter stages of growth, both as soluble and membrane-bound enzymes. When grown in the presence of high concentrations of organic phosphates, the synthesis and expression of AcP and AlP was unaltered. Cyto- and immuno-chemical localization situated the phosphatases in the periplasmic space, at the cell surface, and in membranous vesicles.     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

Rat intestinal brush border membrane peptidases. II. Enzymatic properties, immunochemistry, and interactions with lectins of two different forms of the enzyme.

The properties of two purified peptidases derived from the intestinal brush border membrane of the rat have been investigated. The pH optima, heat stabilities, substrate specificities, and metal ion requirements of the two enzymes and the effects of inhibitors on their activities were nearly identical. The isoenzymes catalyzed the hydrolysis of a wide range of peptides containing from 2 to 8 amino acid residues. The enzymes are aminopeptidases; no evidence for carboxypeptidase or endopeptidase activity was found. For hydrolysis, there appears to be an absolute requirement for an L-amino acid at the NH2-terminus of the peptide substrate. There was a similar but less absolute requirement for the penultimate NH2-terminal amino acid. Thus, although peptides of the type L-aminoacyl-L-proline, L-aminoacyl-L-prolyl-(L-amino acid)n, or L-aminoacyl-D-amino acid were not hydrolyzed, L-leucyl-beta-naphthylamide could be utilized as a substrate. The enzymes appeared to be metalloenzymes in that metal ion-chelating agents could inhibit their activities. Co2+ partially restored the activities lost by chelation. Immunodiffusion studies showed that the two enzymes were immunologically identical. The antipeptidase antisera were specific for the enzymes and did not react with other constituents of the intestinal cell. Both enzymes have binding sites for the lectin phytohemagglutinin which recognizes N-acetylgalactosamine residues located at or near the terminal positions of glycoprotein carbohydrate chains. Both the lectin and the antibodies inhibited enzyme activities, but the mechanisms of inhibition appeared to be different.     

Characterization of Membrane-Bound and Soluble Catechol-O-Methyltransferase from Human Frontal Cortex

Abstract: Catechol- O -methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane-bound form. Attempts to solubilize the membrane-bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X-100 was capable of solubilizing membrane-bound COMT suggested that the membrane-bound transferase is an integral membrane protein. The membrane-bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH-activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl₂, both enzymes displaying 65% inhibition at 2.5 m M CaCl₂. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed K _i values for the membrane-bound transferase five- to 10-fold lower than those observed for the soluble transferase. Solubilization of membrane-bound COMT in Triton X-100 resulted in an increase in the apparent K _m value of the membrane-bound transferase for dopamine. The increase in K _m appeared to be due to apparent competitive inhibition by Triton X-100 and reached a limiting value of approximately 80 μM. These results confirm that membrane-bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.     

Maize leaf proteases and their effect on endogenous inorganic pyrophosphatase

Several different proteolytic enzymes are present in leaf and root tissue of maize seedlings. The activity of these enzymes diminishes to a basal level by the time seedling height reaches 20–30 cm. We have partially characterized an endopeptidase with trypsin-like specificity and two aminopeptidases, all from leaf tissue, and compared them to previously reported proteases from maize. Both the endopeptidase and the aminopeptidases degrade the maize leaf enzyme, inorganic pyrophosphatase. Modification of the pyrophosphatase by the peptidases results in the formation of catalytically active, electrophoretically distinct products. The aminopeptidases have little effect on several other maize leaf enzymes, but also modify yeast inorganic pyrophosphatase.     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.     

Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Purification and characterization of two distinct dipeptidyl aminopeptidases in soluble fraction from monkey brain and their action on enkephalins

Two distinct dipeptidyl aminopeptidases, which were designated DPP-A and DPP-B, were purified from soluble fraction of monkey brain using Leu-enkephalin as the substrate. The enzymes were purified 187 and 136 fold, respectively. Both enzymes showed the optimum pH in neutral range. Their molecular weights were almost equal and were estimated to be about 100,000. Their Km values with Leu-enkephalin as the substrate were 5.6 X 10(-5) and 1.1 X 10(-5) M, respectively. Among synthesized substrates, the highest affinity of the enzymes was toward arginyl-arginine beta-naphthylamide with the Km values of 6.25 X 10(-5) and 6.41 X 10(-5) M, respectively. Both enzyme activities were inhibited by the metal-chelators DFP and PCMB. Two hundred fifty microM arphamenine A inhibited DPP-A and -B with inhibition of 36.6% and 44.1%, respectively. Beta-endorphin, ACTH, and glucagon inhibited only DPP-B, while beta-lipotropin and angiotensin II inhibited both DPP-A and -B when Leu-enkephalin was used as the substrate.