Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

Purification and characterization of human erythrocyte uridylyl transferase

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.     

Purification and characterization of activated human erythrocyte prolidase

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 degrees C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58,000 when measured by SDS-PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58,000.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.     

Functions of extracellular lysine residues in the human erythrocyte anion transport protein

The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.     

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Purification and characterization of the N-terminal propeptide of human type III procollagen.

The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.     

Purification and characterization of chicken erythrocyte ferrochelatase.

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.     

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

Monoclonal antibodies against human erythrocyte band 3 protein. Localization of proteolytic cleavage sites and stilbenedisulfonate-binding lysine residues

Monoclonal antibodies against the membrane domain of human red blood cell band 3 protein have been prepared and used in topographical studies of the arrangement of the polypeptide in the membrane. One of the antibodies binds to a site near the N terminus of the membrane domain; another binds to a site near the C terminus. The latter has been used to localize a site of intracellular trypsin digestion. The cleavage site, in human band 3, corresponds to Lys-761 in mouse band 3; the site is 168 residues from the C terminus of the protein. This is the first intracellular site in the membrane domain (other than the N terminus) that has been localized in the primary structure. The antibody that binds to the N-terminal portion of the membrane domain has been used to identify a new S-cyanylation cleavage site about 7,000 daltons from the C terminus. Proteolysis/cross-linking experiments with the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) reveal that one end of the H2DIDS reacts covalently with a lysine residue that is between about 70 and 168 residues from the C terminus of band 3. In addition to placing restrictions on the location of the H2DIDS-binding lysine, these studies provide direct evidence that the C-terminal 28,000-dalton papain fragment crosses the membrane at least three times. With previous data on the remainder of the membrane domain, there is now direct evidence that the band 3 polypeptide crosses the membrane at least eight times.     

Model system studies of staining procedures for lysine and arginine residues

Summary Using polyacrylamide films containing poly-lysine, polyargine and DNA as test models, a variety of reportedly specific staining procedures have been examined. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin while arginine will stain with fast green, if proteins containing both amino-acids are stained with the dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acid-arginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrochloric acid in a Feulgen-like procedure which stains DNA to the same level as the classical hydrochloric acid based procedure while also staining arginine present.     

Purification and characterization of a casein kinase from human erythrocyte cytosol

A casein kinase was extracted from human erythrocyte cytosol and purified by ammonium sulfate precipitation, chromatography on DEAE and phosphocellulose, and affinity chromatography on ATP-agarose. This enzyme did not use histone as a substrate; its activity was not stimulated by cyclic nucleotides. The pH of optimal activity was 6.5. The enzyme had an absolute requirement of Mg²⁺ ions at an optimal concentration of 30 mM; activity was stimulated by Na⁺ and K⁺ at a maximal concentration of 0.125 M and inhibited by Ca²⁺. Casein was used as a substrate with a Km of 0.25 mg/ml; ATP was the preferential phosphoryl donor with a Km of 14.7 μM; GTP may be used with a lower yield and a Km of 26.3 μM. ADP was a competitive inhibitor of ATP with a Ki of 14 μM. 2–3 DPG was an allosteric inhibitor of ATP with an apparent Ki of 4.6 mM and a Hill coefficient of 3.8. Kinetic data indicate that the reaction follows a coordinated mechanism with ATP as the first substrate and subsequent formation of a ternary complex with the protein. SDS-PAGE of the purified enzyme showed two different peptide chains of molecular weight 35 000 and 25 000.     

Purification and characterization of two human liver carboxylesterases

1. Two carboxylesterases (EC 3.1.1.1) purified from human livers were distinguished by pI (isoelectric point), nondenaturing polyacrylamide gel electrophoresis, molecular weight, catalytic activity, N-terminus and immunological cross-reactivity. 2. The low pI carboxylesterase has not been reported previously. 3. Numerous bands seen when each enzyme was focused on analytical IEF gels could not be separated. 4. When sections of the band pattern was refocused, the original complete band pattern was generated. 5. Both the mid and low pI carboxylesterases had catalytic activity for xenobiotics as well as medium and long chain fatty acid esters.     

Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme.

The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.     

Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues.

1, 2-Cyclohexanedione reacts specifically with the guanidino group of arginine or arginine residues at pH 8 to 9 in sodium borate buffer in the temperature range of 25-40 degrees. The single product, N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine (DHCH-arginine) is stable in acidic solutions and in borate buffers (pH 8 to 9). DHCH-Arginine is converted to N-7-adipyl-L-arginine by periodate oxidation. The structures of the two compounds were elucidated by chemical and physicochemical means. Arginine or arginyl residues can be regenerated quantitatively from DHCH-arginine by incubation at 37 degrees in hydroxylamine buffer at pH 7.0 FOR 7 TO 8 hours. Analysis of native egg white lysozyme and native as well as oxidized bovine pancreatic RNase, which were treated with cyclohexanedione, showed that only arginine residues were modified. The utility of the method in sequence studies was shown on oxidized bovine pancreatic ribonuclease A. Arginine modification was complete in 2 hours at 35 degrees in borate buffer at pH 9.0 with a 15-fold molar excess of the reagent. The derived peptides showed that tryptic hydrolysis was entirely limited to peptide bonds involving lysine residues, as shown both by two-dimensional peptide patterns and by isolation of the resulting peptides. The stability of DHCH-arginyl residues permits isolation of labeled peptides.     

Purification and partial characterization of an arginine binding molecule from human placenta

A binding molecule for L-arginine has been isolated from human placental membranes and partially characterized. It exhibits specificity for L-arginine almost exclusively with no apparent cooperativity of binding as seen by Scatchard analysis (K_d = 0.36nM). Enzymatic probes indicate a molecule containing important carbohydrate and lipid moieties.