Rationalization of aminopeptidase activities in human skeletal muscle soluble extract

Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were found to be hydrolysed by human skeletal muscle soluble fraction, fractionation of the latter via anion-exchange and gel-filtration chromatography resolved only five types of separable aminopeptidase (with activity relative to alanyl aminopeptidase in parentheses): alanyl aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.14, 100%), arginyl aminopeptidase (two isoenzymes, L-arginyl-L-lysyl)-peptide hydrolase, EC 3.4.11.6, 15%); pyroglutamyl aminopeptidase (5-oxoprolyl-peptide hydrolase, EC 3.4.19.3, 3%); leucyl aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1, 1.5%) and alpha-glutamyl aminopeptidase (0.2%). Thus over 80% of the total aminopeptidase activity (expressed in relative terms) in human skeletal muscle soluble fraction can be accounted for by a single enzyme, the major aminopeptidase. A single peak of activity, which co-eluted with the major aminopeptidase after anion-exchange and gel-filtration chromatography, was obtained after assay with the following aminoacyl-7-amino-4-methylcoumarin derivatives: glycyl-, isoleucyl-, lysyl-, methionyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. Thus, the hydrolysis of these derivatives by skeletal muscle soluble fraction occurs principally via the major aminopeptidase and not by specific enzymes, as previously suggested (Wada and Aoyagi, 1983). These results illustrate the difficulty in measuring individual aminopeptidase activities in muscle homogenate and soluble fraction, and the danger in ascribing apparent aminopeptidase activity to 'specific' enzymes.     

Leucine aminopeptidase (bovine lens). Effect of pH on the relative binding of Zn2+ and Mg2+ to and on activation of the enzyme.

Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.     

Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.     

Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.     

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.     

Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2

Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.     

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

Cloning, chromosomal sublocalization of the human soluble aminopeptidase P gene (XPNPEP1) to 10q25.3 and conservation of the putative proton shuttle and metal ligand binding sites with XPNPEP2

Human soluble ("cytosolic") aminopeptidase P (hsAmP) is an aminoacylprolyl hydrolase (EC 3.4.11.9) present in all tissues yet examined. hsAmP is related in terms of catalytic specificity to an ectoenzyme, membrane aminopeptidase P (hmAmP), which is largely limited in distribution to endothelia and brush border epithelia. Although both enzymes can degrade oligopeptides having N-terminal Xaa-Pro- moieties, hsAmP and hmAmP are of relatively low sequence homology. Recently, it has been shown that the two enzymes are not products of splice variants of the same gene. How hsAmP relates to hmAmP has clinical significance in that both can inactivate bradykinin, and AmP deficiency states have been described. The hmAmP gene (XPNPEP2) is disposed at chromosome Xq25, a disposition with clear meaning in terms of inheritance of hmAmP deficiencies. To further explore similarities and differences between hsAmP and hmAmP, the present study was begun to determine the chromosomal disposition of the hsAmP gene. Here we show that the gene is sublocalized on chromosome 10q25.3. We also show that hsAmP and hmAmP contain homologous blocks of sequence common to members of the "pita bread-fold" protein family, of which Escherichia coli methionine aminopeptidase is the prototype. The prototype is known to contain a proton shuttle and five divalent metal ligands, counterparts of which we identify in the homologous blocks of sequence in both hsAmP and hmAmP and compare to E. coli aminopeptidase.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Association-dissociation of the flavoprotein hog kidney D-amino acid oxidase. Determination of the monomer-dimer equilibrium constant and the energetics of subunit association.

The enzyme concentration dependence of spectrophotometric titrations of hog kidney D-amino acid oxidase [EC 1.4.3.3] with p-aminobenzoate was studied. The monomer-dimer equilibrium constant of the oxidized holoenzyme at 25 degrees C was estimated to be 7 X 10(5)M-1 at pH 7.5 and 4X 10(6)M-1 at pH 8.3. The energetics of subunit association are discussed.     

Purification and characterization of guinea pig serum aminoacylproline hydrolase (aminopeptidase P).

Aminoacylproline hydrolase (EC 3.4.11.9) of guinea pig serum has been obtained as two apparently homogeneous isoforms. Dialyzed serum was chromatographed successively on Affi-gel blue, hydroxyapatite, DE-cellulose, phenyl-Sepharose, an affinity matrix for angiotensin converting enzyme and concanavalin-Sepharose. On the latter matrix, 68% of the enzyme activity was eluted with alpha-methyl mannoside at 10 and 100 mM, and 29% was eluted with alpha-methyl glucoside, 500 mM, at 56 degrees C. The two fractions ('biantennary' and 'high mannose' fractions, respectively) were concentrated and then chromatographed separately on Sephacryl S-200HR. Both fractions were eluted as expected for a globular protein of Mr 217,000. On SDS-PAGE, under reducing and non-reducing conditions, each of the concanavalin-Sepharose fractions was separated into two protein bands, Mr 89,000 and Mr 81,500. Each of the bands was found to be N-blocked when N-terminal amino acid sequencing was attempted. The reaction of the 'biantennary' fraction with the synthetic substrate Arg-Pro-Pro-[3H]benzylamide was characterized in part: Km 0.7 microM, kcat 124.6 min-1, kcat/Km 1.78.10(8) M-1 min-1. Hydrolysis of the substrate was strongly inhibited by bradykinin and those of its lower homologs that contain two adjacent proline residues. Cu2+ was strongly inhibitory. Co2+ at 30 microM activated the enzyme, as did Mn2+, Mg2+ and Ca2+ at higher concentrations. Sulfhydryl compounds, including captopril, inhibited the enzyme as did 1,10-phenanthroline. Iodoacetamide and N-ethylmaleimide had no effects, but 4-hydroxymercuribenzoate conferred a partial inhibition over a remarkably wide concentration range: 0.34-1400 microM. Amastatin and bestatin did not inhibit the enzyme. Aminoacylproline hydrolase of guinea pig serum appears to be a heterogeneous, glycosylated metallo-enzyme with a high affinity for bradykinin and related peptides in which the sequence Pro-Pro, Xaa-Pro-Pro or Xaa-Pro-Hyp is N-terminal.     

Binary interactions of troponin subunits

The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.     

Yeast aminopeptidase I. Chemical composition and catalytic properties.

An aminopeptidase (alpha-aminoacyl L-peptide hydrolase, EC 3.4.11.1) was purified to homogeneity from autolysates of brewer's yeast. The enzyme which is responsible for most of the yeast cell's aminopeptidase activity is a glycoprotein containing about 12% of conjugated carbohydrate and 0.02% Zn2+ and having a complex quaternary structure. The active species has a molecular weight of approx. 600000 and an isoelectric point of 4.7. The enzyme is remarkably stable, even in dilute solutions. All types of L-amino acid and peptide derivatives containing a free amino terminus are attacked, including amino acid amides and esters. As to its substrate specificity, the enzyme belongs to the so called leucine-aminopeptidases. It is strongly and specifically activated by Zn2+ and Cl- (or Br-) and inactivated by metal-chelating agents. The activation by Zn2+ seems to be mediated by a conformational transition which affects exclusively V and leads to a form of the enzyme which enhanced stability against heat. Halide anions, on the other hand, are acting as positive allosteric effectors, modulating both V and Km.     

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.     

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.