Biochemical characterization of a catalase inhibitor from maize

Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms.     

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.     

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Inhibition of human factor IXa by human antithrombin.

A procedure is presented for the purification of Factor IX from human plasma. The final product is homogeneous as judged by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Furthermore, it is completely free of other coagulation component activities. Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IXa in the presence of calcium ions. This activated species is added to purified antithrombin-heparin cofactor and the interaction is studied in the presence and absence of heparin. Antithrombin-heparin cofactor is found to be a progressive, time-dependent inhibitor of Factor IXa and neutralizes approximately 57% of this enzyme's proteolytic activity within 30 min. The addition of heparin dramatically accelerates the rate of this interaction with virtually complete inhibition of Factor IXa occurring within 15 s. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin-heparin cofactor functions as a potent inhibitor of Factor IXa by forming an undissociable complex with the enzyme which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.     

An acetylcholine receptor preparation lacking the 42,000 Dalton component.

Acetylcholine receptor has been purified from $\text{Electrophorus}$ in the presence of the serine protease inhibitor phenylmethyl sulfonyl flouride. The purified material has a specific toxin-binding capacity of 3.6 nmoles of toxin per mg of protein. Electrophoresis of reduced, dissociated receptor on acrylamide gels containing sodium dodecyl sulfate reveals components of 110,000, 60,000, 54,000, and 48,000 daltons. No component with an apparent molecular weight of less than 48,000 daltons is seen. Limited digestion of this preparation with trypsin results in the appearance of components of 44,000 and 42,000 daltons. Prolonged digestion with trypsin generates species with apparent molecular weights of less than 42,000 and has no effect on the specific protectable toxin-binding capacity of the preparation.     

Purification and properties of an endothelial cell growth factor from human platelets

An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin.     

Purification and characterization of protease inhibitors from urine during pregnancy (author's transl)

Two forms of urinary trypsin inhibitor, A and B, were purified from the pooled urine from pregnant women using non-denaturing methods. The inhibitor B arose from the inhibitor A and was not present in native urine. Electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate indicated a new heterogeneity of the inhibitor B with molecular weights of 33 000 and 24 000; the molecular weight obtained for the inhibitor A was 50 000. Inhibitors A and B were acidic proteins with an isoelectric pH of about 2.6 for A and about 4.2 for B. Inhibitor A and inter-alpha-trypsin inhibitor formed a precipitate with an antiserum to purified inhibitor B. But neither inhibitor A nor inhibitor B formed a precipitate with anti whole human serum or anti-inter-alpha-trypsin inhibitor antiserum. Measurements of specific activity of inhibitor A were consistent with two active sites in the molecule.     

Purification and characterization of bovine tissue factor

Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain. The protein is an integral membrane glycoprotein with an apparent molecular weight of 43,000 as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The apoprotein was first purified by extraction with Triton X-100 and repeated preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Antiserum was produced against a few micrograms of purified apoprotein and was used to construct an immunoadsorbent column. The column was then used for affinity purification of the apoprotein directly from the Triton X-100 extract, thereby significantly increasing the amount of purified protein produced. The purification scheme may be generally useful for the rapid and large scale purification of membrane proteins. Tryptic digestion of the apoprotein in Triton X-100 cleaved a peptide of approximately 3000 daltons without affecting the activity. The activity was recovered directly from stained SDS polyacrylamide gels, and the profile of recovered activity corresponded directly with the stained bands. The activity shifted along with the protein band following tryptic digestion, thus demonstrating that the protein observed on the gels is tissue factor. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipid. Optimal activity was observed at phospholipid to protein ratios (w/w) greater than 450:1.     

An inhibitor of plasminogen activation from human placenta. Purification and characterization

Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.     

Purification and properties of 16 alpha-hydroxyprogesterone dehydroxylase from Eubacterium sp. strain 144

Eubacterium sp. strain 144 converts 16 alpha-hydroxyprogesterone to 17-isoprogesterone. The first step of this reaction is catalyzed by 16 alpha-hydroxyprogesterone dehydroxylase (16 alpha-dehydroxylase). This enzyme was purified 40-70-fold and characterized. 16 alpha-Dehydroxylase was found to be active in two molecular weight forms of Mr 181 000 and 326 000. A subunit relative molecular weight of 42 400 was determined by sodium dodecyl sulfate gel electrophoresis of the purified enzyme. Although active with both 16 alpha-hydroxyprogesterone and 16 alpha-hydroxypregnenolone, the affinity of 16 alpha-dehydroxylase for the latter steroid was twice that of the former based on the apparent Km values. Evidence of possible substrate inhibition at high concentrations was seen with 16 alpha-hydroxypregnenolone. 16-Ketoprogesterone was found to be a competitive inhibitor of 16 alpha-dehydroxylase with respect to both steroid substrates. Although generally unaffected by low concentrations of non-ionic detergents, 16 alpha-dehydroxylase activity was stimulated 3-7-fold by sodium dodecyl sulfate and inhibited strongly by cetyltrimethylammonium bromide.     

Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells

CND41 is a 41 kDa DNA-binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate-labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2-4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe(3+) and sodium dodecyl sulfate.     

Creatine amidinohydrolase of Pseudomonas putida: crystallization and some properties.

Creatine amidinohydrolase (EC 3.5.3.3, creatinase) of Pseudomonas putida var. naraensis C-83 was purified by column chromatography on sarcosine-hexamethylenediamine-Sepharose and Sephadex G-200 and then crystallized in the presence of ammonium sulfate. The purified preparation appeared homogeneous on disc gel electrophoresis and ultracentrifugal analysis. It was most active at pH 8 and showed a K_m value of 1.33 mm for creatine. Estimation of the molecular weight by the meniscus depletion method yielded a value of 94,000. A value of 47,000 was obtained, however, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is composed of two subunits. Inhibition experiments suggested that a sulfhydryl group is closely related to the creatinase activity.     

Heat-stable calmodulin-binding protein in rat testis. Inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase activity

An inhibitor of procine brain calmodulin-dependent cyclic nucleotide phosphodiesterase was purified about 940-fold from rat testis. This inhibitor inhibited the calmodulin-induced activation of the enzyme without affecting its basal activity. The inhibitor activity was counteracted by a high concentration of calmodulin, but was not by a high concentration of Ca2+. The analysis on polyacrylamide disc gel electrophoresis demonstrated that the inhibitor and calmodulin form a complex in the presence of Ca2+ but not in the presence of excess amount of EGTA. This inhibitor also inhibited the calmodulin-induced activation of Ca2+, Mg2+ -ATPase of human erythrocytes. The inhibitor appeared to be a heat-stable protein, since the inhibitor activity was not attenuated by boiling up to 9 min but was completely abolished by tryptic or chymotryptic digestion. The molecular weights of the inhibitor determined by linear polyacrylamide gradient gel electrophoresis under nondenaturing conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 40,000 and 32,000, respectively. Thus, the inhibitor is suggested to be a calmodulin-binding protein composed of a monomer which has unique properties different from those of other tissues.     

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Purification and properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes

Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase has been purified approximately 3000-fold from bovine brain microsomes by detergent solubilization followed by ion-exchange and affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single protein of molecular weight 43,000. The specificity of the purified enzyme was studied by measuring the catalytic activity with various lysophospholipids and acyl-CoA derivatives. Of the lysophospholipids tested, only lysophosphatidylcholine was a substrate. Less specificity was exhibited toward the acyl-CoA derivatives, although the enzyme showed a clear preference for arachidonoyl-CoA and little or no activity with palmitoyl-CoA or stearoyl-CoA. High concentrations of arachidonoyl-CoA inhibited the enzyme. The velocity was a sigmoidal function of the concentration of lysophosphatidylcholine (LPC) with little activity obtained below 20 microM LPC. The specificity and kinetic properties of the enzyme were altered, however, by incorporation of the enzyme into liposomes composed of a mixture of phospholipids. Decanoyl-CoA and myristoyl-CoA, which were effective substrates for the soluble enzyme, did not serve as acyl donors for the liposome-bound acyltransferase. Furthermore, the liposome-bound enzyme, in contrast to the soluble form of the enzyme, was active at concentrations of LPC below the critical micelle concentration. The liposome-bound enzyme was also substantially less susceptible to thermal denaturation and proteolytic digestion. This modulation of the acyltransferase activity by interaction with phospholipids may relate to the kinetic properties and the regulation of the enzyme in vivo.     

Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases

Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.     

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.