Ascaris suum and Onchocerca volvulus: S-adenosylmethionine decarboxylase

Putrescine-dependent S-adenosylmethionine decarboxylase (EC 4.1.1.50) was demonstrated in Ascaris suum and Onchocerca volvulus; activation was found to be about fourfold by putrescine. Mg2+ did not affect the enzyme activity. A. suum was taken as a model nematode and its S-adenosylmethionine decarboxylase was partially purified and characterized. The molecular weight was estimated to be 220,000. The apparent Km-value for adenosylmethionine was determined to be 17 microM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent Ki-values were found to be 0.24 microM and 0.11 microM, respectively. The dependence of filarial worms on uptake and interconversion of putrescine and polyamines as well as properties of the S-adenosylmethionine decarboxylase, different from the host enzyme, points to the polyamine metabolisms as a useful target for chemotherapy.     

Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.     

Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn²⁺ ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Purification of S-adenosylmethionine: epsilon-N-L-lysine methyltransferase. The first enzyme in carnitine biosynthesis.

The initial steps of carnitine biosynthesis in Neurospora crassa involve the methylation of the epsilon-amino group of lysine as follows: Lysine A leads to monomethyllysine B leads to dimethyllysine C leads to trimethyllysine. The methyl donor is S-adenosylmethionine. An enzyme, S-adenosylmethionine:epsilon-N-L-lysine methyltransferase, has been purified from N. crassa to near homogeneity as judged by column chromatography, polyacrylamide gel electrophoresis, and ultracentrifugation. This protein catalyzes all three methylation reactions. The reaction rates are: A less than B less than C. Sedimentation equilibrium and molecular filtration give a molecular weight of 22,000 for the protein. Sedimentation equilibrium analysis of the protein in 6 M guanidine hydrochloride and sodium dodecyl sulfate-polyacrylamide gel electrophoresis do not detect the possibility of subunit structure. The enzyme contains no half-cystine but does contain several acidic residues. The protein exhibits an absorption band between 400 and 420 nm which is 40 to 50 times less than the absorption seen at 280 nm and is not affected by the presence of substrates. The source of this absorption in unkown.     

Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus

A new hydrolase for conjugated bile acids, tentatively named chenodeoxycholyltaurine hydrolase, was purified to homogeneity from Bacteroides vulgatus. This enzyme hydrolyzed taurine-conjugated bile acids but showed no activity toward glycine conjugates. Among the taurine conjugates, taurochenodeoxycholic acid was most effectively hydrolyzed, tauro-beta-muricholic and ursodeoxycholic acids were moderately well hydrolyzed, and cholic and 7 beta-cholic acids were hardly hydrolyzed, suggesting that this enzyme has a specificity for not only the amino acid moiety but also the steroidal moiety. The molecular weight of the enzyme was estimated to be approximately 140,000 by Sephacryl S-300 gel filtration and the subunit molecular weight of the enzyme was 36,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH was in the range of 5.6 to 6.4. The NH2-terminal amino acid sequence of the enzyme was Met-Glu-Arg-Thr-Ile-Thr-Ile-Gln-Gln-Ile-Lys-Asp-Ala-Ala-Gln. The enzyme was activated by dithiothreitol, but inhibited by sulfhydryl inhibitors, p-hydroxymercuribenzoate, N-ethylmaleimide, and dithiodipyridine.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans

Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the , high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.     

Quantitation of S-adenosylmethionine decarboxylase protein

A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32 000, (b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigen had a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in the amount of enzyme protein measured both by titration of the active site and by radioimmunoassay.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.     

Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema

The leukotriene A(4) hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A(4) to leukotriene B(4) and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B(4). Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A(4) hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.     

Interaction of constituent subunits in ribulose 1,5-bisphosphate carboxylase from Aphanothece halophytica

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) from the halophilic cyanobacterium, Aphanothece halophytica, dissociates into catalytic core (large subunit A oligomer) and small subunit B under low ionic strength during sucrose density gradient centrifugation. Supplementation of KCl, NaCl, or K2SO4 ( [I] = 0.3 M) partly prevents the dissociation, the preventive effect of divalent cation salts such as MgCl2 and CaCl2 being more effective than monovalent cation salts. RuBisCO with its higher-plant-type molecular form can be isolated from the cyanobacterial extracts using gradient medium containing 0.3 M KCl, 20 mM MgCl2, and 10 mM CaCl2. The isolated enzyme contains large subunit A and small subunit B in a molar ratio of approximately 1:1, estimated from the densitometric scanning of Coomassie blue-stained gels. During the second sucrose density gradient centrifugation to remove minor contaminants, a small amount of subunit B is depleted from the holoenzyme. Determination of the molecular weight by equilibrium centrifugation and electron microscopic observation have confirmed that the cyanobacterial RuBisCO has an A8B8-type structure. The enzyme activity per se is found to be sensitive to concentrations of salts, and small subunit B is obligatory for the enzyme catalysis. It has been shown that the more the enzyme activity is inhibited by salts, the tighter the association of subunit B becomes. It is likely that the active enzyme retains the loose conformational structure to such an extent that the dissociable release of subunit B from the holoenzyme in vivo is not allowed.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

Expression and characterization of recombinant human acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides

The molecular cloning and eukaryotic cell expression of the complementary DNA for human neutrophil acyloxyacyl hydrolase (AOAH) are described. AOAH is a leukocyte enzyme that selectively removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (endotoxins), thereby detoxifying the molecules. The two disulfide-linked subunits of the enzyme are encoded by a single mRNA. The amino acid sequence of the protein contains a lipase consensus sequence in the large subunit and a region in the small subunit that is similar to the saposins, cofactors for sphingolipid hydrolases. The recombinant enzyme, like native AOAH, hydrolyzes secondary acyl chains from more than one position on the lipopolysaccharide backbone. Acyloxyacyl hydrolase is a novel two-component lipase that, by deacylating lipopolysaccharides, may modulate host inflammatory responses to Gram-negative bacterial invasion.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

Purification and Some Properties of a Membrane-Bound Aminopeptidase A from Streptococcus cremoris

A membrane-bound l-alpha-glutamyl (aspartyl)-peptide hydrolase (aminopeptidase A) (EC 3.4.11.7) from Streptococcus cremoris HP has been purified to homogeneity. The free gamma-carboxyl group rather than the amino group of the N-terminal l-alpha-glutamyl (aspartyl) residue appeared to be essential for catalysis. No endopeptidase activity could be established with this enzyme. The native enzyme is a polymeric, most probably trimeric, metalloenzyme (relative molecular weight, approximately 130,000) which shows on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels apparent high relative molecular weight values due to (lipid?) material dissociable with butanol. The subunit (relative molecular weight, approximately 43,000) is catalytically inactive. The enzyme is inactivated completely by dithiothreitol, chelating agents, and the bivalent metal ions Cu and Hg. Of the sulfhydryl-blocking reagents tested, only p-hydroxymercuribenzoate appeared to inhibit the enzyme. Activity lost by treatment with a chelating agent could be restored by Co and Zn. The importance of the occurrence of an aminopeptidase A in S. cremoris with respect to growth in milk is discussed.     

S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization

S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.     

L-deaza-5'-noraisteromycin

(+/-)-1-Deazaaristeromycin (4) has been reported to be an inactivator of S-adenosylhomocysteine (AdoHcy) hydrolase and, as a consequence, to affect S-adenosylmethionine (AdoMet) mediated macromolecular biomethylations. To extend this to our program focused on 5'-noraristeromycin derivatives as inhibitors of the same hydrolase enzyme as potential antiviral agents, both enantiomers of 1-deaza-5'-noraristeromycin (5 and 20) have been prepared. Compounds 5 and 20 were evaluated against the following viruses: vaccinia, cowpox, monkeypox, Ebola, herpes simplex type 1 and 2, human cytomegalovirus, Epstein Barr, varicella zoster, hepatitis B, hepatitis C, HIV-1 and HIV-2, adenovirus type 1, measles, Pichinde, parainfluenza type 3, influenza A (H1N1 and H3N2), influenza B, Venezuelan equine encephalitis, rhinovirus type 2, respiratory syncytial, yellow fever, and West Nile. No activity was found nor was there any cytotoxicity to the viral host cells.     

Characterizations of acylagmatine amidohydrolase and carboxypeptidase from Fusarium anguioides.

Previously an enzyme, named acylagmatine amidohydrolase, hydrolyzing bleomycin B2 to bleomycinic acid and agmatine was found in the mycelia of Fusarium anguioides Sherbakoff. In this work the enzyme was purified further, but not completely. The crude enzyme preparation hydrolyzed various acylagmatines and also peptidyl arginine, but the latter activity could be separated from acylagmatine amidohydrolase activity by gel filtration on Sephadex G-100. The enzyme was inhibited by PCMB and its molecular weight was estimated as 65,000 by gel filtration. It showed substrate specificity with respect to the alkyl-chain length of the amine moiety. The other hydrolase fraction with activity toward Bz-Gly-Arg was found to be of a sort of carboxypeptidase, which preferentially hydrolyzed peptides with arginine or lysine at the carboxyl terminus, including bradykinin, but liberated neutral amino acids as well from the terminus when the penultimate residue of the substrates was phenylalanine. With Bz-Gly-Arg as substrate Fusarium carboxypeptidase was sensitive to chelating agents but not to diisopropyfluorophosphate, and its molecular weight was estimated to be 145,000.