EDTA Inhibition Research Articles

Flow-injection/standard subtraction method for the determination of iron(III) based on its catalytic effect and inhibition of EDTA

A new approach for the flow-injection catalytic determination of 10 −6 M levels of iron(III) is proposed. This method is based on the catalytic effect of iron(III) on the oxidative coupling of p-anisidine with N,N-dimethylaniline to form a blue compound ( λ max=735 nm) in the presence of hydrogen peroxide and 1,10-phenanthroline as an activator, and the inhibitory effect of EDTA on the iron(III)-catalyzed reaction. The concentration of iron(III) can be determined from only two flow signals which are obtained by injecting the iron(III) and iron(III) plus EDTA solutions into the flow lines without a calibration graph for iron(III); this procedure may be called standard subtraction. The proposed method permits the analysis of 12 samples per hour (24 peaks per hour) and was successfully applied to the determination of iron in standard iron ore samples.

Gelatinolytic activity in tracheal epithelial lining fluid and in blood from horses with chronic obstructive pulmonary disease

SUMMARY Objectives To determine whether gelatinolytic activity in tracheal epithelial lining fluid (TELF), blood neutrophils, and blood lymphocytes from horses was metalloprotease activity, and to compare, for healthy horses and horses with chronic obstructive pulmonary disease, gelatinolytic activity in neutrophils, lymphocytes, and serum with activity in TELF. Animals 7 horses with chronic obstructive pulmonary disease (COPD) and 4 healthy control horses. Procedure Neutrophils and lymphocytes were obtained by means of Percoll separation. Zymography was used to detect gelatinolytic activity; EDTA inhibition and 4-aminophenyl mercuric acetate activation were used to verify that gelatinolytic activity was metalloprotease activity. Results Gelatinolytic activity was significantly higher in TELF from horses with COPD than in TELF from healthy horses. For all samples, EDTA inhibited and APMA activated gelatinolytic activity. Gelatinolytic activity of neutrophils, lymphocytes, and serum was not significantly different between healthy horses and horses with COPD. Conclusions Results suggested that gelatinolytic activity in TELF from horses is metalloprotease activity. Gelatinolytic activity is increased in TELF from horses with COPD, but not in serum, neutrophils, or lymphocytes. Neutrophils and lymphocytes are possible sources of gelatinolytic activity in TELF. Clinical Relevance Measurements of serum, blood neutrophil, or blood lymphocyte gelatinolytic activity were of little value in distinguishing horses with COPD from healthy horses. (Am J Vet Res 1998;59:818–823)

In vitro study of the NS2-3 protease of hepatitis C virus.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.

Purification and Characterization of Blood Group A-degrading Isoforms of α-N-Acetylgalactosaminidase from Ruminococcus torques Strain IX-70

To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized alpha-N-acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torques strain IX-70. Three isoforms separated during hydrophobic interaction chromatography. Hydroxyapatite chromatography further resolved the most hydrophilic, isoform I, into isoforms IA and IB. The most hydrophobic, isoform III, differed from IA and IB by a more acidic pH optimum, greater heat resistance, greater sensitivity to alkylating agents, and anomalous retardation during gel filtration chromatography. Isoform IB differed from IA and III in N-terminal amino acid sequence and in sensitivity to EDTA inhibition. Each cleaved nonreducing alpha(1-->3)-N-acetylgalactosamine residues from human blood group A and AB mucin glycoproteins, Forssman hapten, and blood group A lacto series glycolipids. The apparent molecular mass of denatured isoform subunits of IA, IB, and III-PII (158, 173, and 201 kDa, respectively) bore no integer relationship to the apparent molecular mass of the native isoforms (265, 417, and 530 kDa), but the latter bore a ratio of 1.96:3.09:3.93 to the weight-average apparent molecular mass of native IA (135 kDa), suggesting that the isoforms are multimers of a 135-kDa sequence. Isoforms IA and III-PII had an identical N-terminal amino acid sequence which showed homologies to the N-terminal sequence of sialidases produced by Bacteroides fragilis SBT3182, another commensal enteric bacterium.

Covalent Cross‐linking Effects on Thermo‐Rheological Profiles of Fish Protein Gels

ABSTRACTThe extent of covalent cross‐linking in surimi sols by transglutaminase was modified by addition of either EDTA (inhibition by calcium sequestering), variation of preincubation time at 25°C to allow action of endogenous transglutaminase, or by addition of a microbial transglutaminase. Gels were cooked following preincubation to fix the effects of the cross‐linking reaction. Torsion, dynamic (small strain) test and stress relaxation measurements were made on the gels over a range of temperatures. Gels preincubated with EDTA had similar rheological response to temperature as gels cooked without preincubation. The stress relaxation and thermo‐rheological profiles of gels with increased covalent cross‐linking (induced by added or endogenous transglutaminase) were increasingly rubber elastic in their behavior.

Lymphocyte adhesive interactions with cultured parotid salivary gland epithelial cells from rats.

Cellular interactions control lymphocyte localization within salivary gland tissues and contribute to the immune defense of oral surfaces. We examined lymphocyte adherence to cultured parotid cells using an in vitro assay and found good correlation with previously reported binding to parotid gland frozen sections. Thoracic duct lymphocytes (TDL) bound to parotid cells in greater numbers than thymocytes (74 vs 11 cells/mm2). B cells showed preferential adherence compared to T cells (75% vs 28%). TDL binding was inhibited by sodium azide or cytochalasin B (60% and 80%, respectively). EDTA inhibition (63%) was restored by replacing calcium (9%) but not magnesium (65%). Binding was inhibited by fucoidin or phosphomannan (approximately 70%). Fibronectin peptides had no effect. Culture supernatants were inhibitory for TDL adherence (60%), suggesting that molecules involved in lymphocyte localization may be shed and that parotid cell cultures will be useful for ligand isolation and characterization.

Integrin alpha 4 cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to alpha 4/180 formation.

Here we describe experiments in which we mutated four of the six integrin alpha 4 subunit cysteine residues that are not present in most other integrin alpha subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin alpha 4 beta 1 and/or to CS1 peptide required the presence of both alpha 4 Cys 278 and Cys 717. In addition, optimal ligand binding required divalent cations and reduced cysteines, as evidenced by EDTA and N-ethylmaleimide inhibition results. In a control experiment, an alpha 4 mutation that completely eliminated the alpha 4 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys 278 an Cys 717 mutations markedly altered ligand binding, they had no adverse effect on cell adhesion. Thus, compared with cell adhesion, ligand binding is a distinct and apparently more stringent test of VLA-4 integrin-ligand interactions. In addition, we have established that the formation of the previously described alpha 4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028-2035] also requires Cys 278 and Cys 717, divalent cations and reduced cysteines. thus alpha 4/180 appears to be more functionally relevant than alpha 4/150.

The interaction of methanol dehydrogenase and its cytochrome electron acceptor.

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial 'docking' of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the 'docked' cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

Mechanism of inhibition of horseradish peroxidase-catalysed iodide oxidation by EDTA.

EDTA inhibits horseradish peroxidase (HRP)-catalysed iodide oxidation in a concentration and pH-dependent manner. It is more effective at pH 6 than at lower pH values. A plot of log Kiapp. values as a function of pH yields a sigmoidal curve from which a pKa value of 5.4 can be calculated for an ionizable group on the catalytically active HRP for EDTA inhibition. Among the structural analogues of EDTA, tetramethylethylenediamine (TEMED) is 80% as effective as EDTA, whereas the EDTA-Zn2+ chelate and EGTA are ineffective. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation. Spectral studies show that EDTA can quickly reduce compound I to compound II, but reduction of preformed compound II to the native enzyme is relatively slow, as demonstrated by the time-dependent spectral shift from 417 nm to 402 nm through an isosbestic point at 408 nm. Under steady-state conditions, in a reaction mixture containing HRP, EDTA and H2O2, the enzyme remains in the compound-II form, with absorption maxima at 417, 527 and 556 nm. Direct evidence for one-electron oxidation of EDTA by HRP intermediates is provided by the appearance of an e.s.r. signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-EDTA radical adduct [aN (hyperfine splitting constant) = 1.5 mT] in e.s.r. studies. The signal intensity, however, decreases in the presence of iodide. The KD of the HRP-EDTA complex obtained from optical difference spectroscopy increases with an increase in iodide concentration, and the double-reciprocal plot for EDTA binding indicates that EDTA and iodide compete for the same binding site for oxidation. We suggest that EDTA inhibits iodide oxidation by acting as an electron donor.

Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane.

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.

Purification and properties of membrane-bound 5'-nucleotidase from black rockfish (Sebastes inermis) muscle

A membrane-bound 5'-nucleotidase was purified from black rockfish Sebastes inermis white muscle to homogeneity using Triton X-100 for the solubilization and two steps of affinity chromatography on Con A and 5'-AMP Sepharose. The enzyme is a glycoprotein, and its active site contains a serine residue. The subunit and native molecular weights are 67 000 and 265 000, respectively. The optimal pH and temperature using IMP as substrate were 8.3 and 45 degrees C, respectively. The enzyme hydrolyzes all nucleoside 5' monophosphates tested but not other phosphate esters examined. The Vmax/Km values indicated that the enzyme has the greatest affinity for AMP of the substrates tested. It was inhibited by phenylmethanesulfonylfluoride, diisopropylfluorophosphate, ATP, ADP, adenosine, o-phenanthroline, and EDTA. The inhibition of EDTA was counteracted by addition of divalent cations. It was inhibited by the lipid antioxidant BHA, at millimolar range, but not by BHT.

Purification and properties of a phosphate-irrepressible membrane-bound alkaline phosphatase from Zymomonas mobilis

The phosphate-irrepressible alkaline phosphatase of the ethanol-producing bacterium Zymomonas mobilis was solubilized from membranes by the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB) and purified by fast protein liquid chromatography. The purified enzyme was a monomeric protein of molecular mass 54 kDa, highly resistant to heat and to ionic strength. The alkaline phosphatase of growing Z. mobilis cells (ZAPase), remained active in the presence of an ethanol concentration as high as 100 g l−1. However, in vitro, the stability of the purified ZAPase was severely affected at low ethanol concentration (7·8 g l−1), showing the importance of the membrane environment in vivo. ZAPase differed from other bacterial alkaline phosphatases by having a higher affinity for the substrate 4-nitrophenylphosphate, a higher K i for phosphate, only a partial reactivation by Zn2+ after EDTA inhibition, and a higher specific activity.

Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis.

Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase.

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.

Inhibition of EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron

Inhibition of EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron

Expression of the Germ Cell Alkaline Phosphatase Gene in Human Choriocarcinoma Cells

Alkaline phosphatase (ALP) in human choriocarcinoma cells (malignant trophoblasts) was characterized by its greater sensitivity to EDTA and L-leucine inhibition as compared with the placental isozyme. In addition, both the fully processed and the nonglycosylated forms of choriocarcinoma ALP migrated faster than the corresponding placental enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Choriocarcinoma cells express a 2.6-kilobase (kb) ALP mRNA unlike normal human placenta which expresses a 2.8-kb ALP mRNA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the steady-state levels of the 2.6-kb choriocarcinoma ALP mRNA, which resulted in an increase in enzyme activity and biosynthesis. S1 nuclease analysis using probes derived from a placental ALP cDNA and ribonuclease protection assays employing probes derived from the germ cell ALP gene demonstrated that choriocarcinoma cells express the germ cell ALP gene. The germ cell ALP gene encodes the placental ALP-like isozyme that is primarily expressed in the thymus, testis, and germ cell tumors. The structures of the internal exons (II-X) of the germ cell ALP gene were determined previously based on their similarity to the placental ALP gene. However, the boundaries of exons I and XI (3' exon) of the germ cell ALP gene were not defined due to sequence divergence between the two genes at the 5' and 3' regions. Ribonuclease protection and primer extension assays demonstrated that exon I of this gene is 119 base pairs in length and that germ cell ALP mRNA contains one major transcription initiation site. The isolation and characterization of germ cell ALP cDNA clones from a butyrate-treated choriocarcinoma cDNA library showed that the germ cell ALP mRNA is 2487 bases in length and exon XI of this gene is 1135 base pairs long.

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: Purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5?-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K(m) value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5?-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5?-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 +/- 0.1 degrees C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5?-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an α-amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for α- and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe 3+, Cd 2+, Pb 2+, Hg 2+, Ni 2+ and Ag 1+ were potent inhibitors whereas Zn 2+, Mg 2+, Mn 2+ and Al 3+ were mild inhibitors. Ca 2+, Ba 2+, Sr 2+ and K + stimulated amylase activity in the order of Ca 2+ > Ba 2+ > Sr 2+ > K +. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both α- as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca 2+ and PCMB inhibition by the addition of glutathione (reduced). The K m for α- and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.

Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).

The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.