Side chain cleavage of some cholesterol esters.

For some time it has been known that the side chain of cholesterol sulfate is cleaved by the cleavage enzyme system present in bovine adrenal mitochondria without prior hydrolysis of the sulfate moiety. In this work, other inorganic esters as well as some organic esters of cholesterol were tested as substrates for this enzyme system. The results revealed that cholesterol nitrate, cholesterol phosphate, and a series of acyl esters of cholesterol can also be cleaved by the enzyme system to their respective pregnenolone derivatives without first being hydrolyzed to cholesterol. The rate of oxidation of the carboxylic acid esters decreased as the size of the acyl groups increased. Cholesterol stearate and cholesterol phosphate were demonstrated to be inhibitors of the side chain cleavage of cholesterol. While digitonin, as might be expected, inhibits the cleavage of cholesterol, it accelerates the oxidation of both cholesterol sulfate and cholesterol nitrate. The results reported in this paper add support to the previously proposed hypothesis that more than one cholesterol side chain cleavage enzyme system exists in adrenal mitochondria.     

Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis.

The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.     

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

A colorimetric determination of inositol monophosphates as an assay for d-glucose 6-phosphate–1l-myoinositol 1-phosphate cyclase

A rapid and convenient chemical assay for the enzyme d-glucose 6-phosphate-1l-myoinositol 1-phosphate cyclase is described. The 1l-myoinositol 1-phosphate formed enzymically was oxidized with periodic acid liberating inorganic phosphate, which was assayed. myoInositol 2-phosphate can be assayed in the same way. Glucose 6-phosphate and other primary phosphate esters gave only very small quantities of inorganic phosphate under the conditions described. The K(m) of the enzyme for d-glucose 6-phosphate, 7.5+/-2.5x10(-4)m, was identical with that measured by the radiochemical method. 2-Deoxy-d-glucose 6-phosphate was a powerful competitive inhibitor, K(i) 2.0+/-0.5x10(-5)m, but was not a substrate for the enzyme.     

Distribution of the sites of alkaline phosphatase(s) activity in vegetative cells of Bacillus subtilis

Sites of alkaline phosphatase activity have been located by an electron microscopic histochemical (Gomori) technique in vegetative cells of a repressible strain SB15 of Bacillus subtilis, derepressed and repressed by inorganic phosphate, and in a mutant SB1004 which forms alkaline phosphatase in a medium high in phosphate. The sites of enzyme activity were revealed as discrete, dense, and largely spherical bodies of varying sizes (20 to 150 nm). Cells of both repressible and repression-resistant strains acted on a wide variety of phosphate esters (p-nitrophenylphosphate, beta-glycerophosphate, adenosine-5'-phosphate, glucose-6-phosphate, glucose-l-phosphate, adenosine triphosphate, and sodium pyrophosphate) to produce inorganic phosphorus under conditions of alkaline phosphatase assay [0.05 m tris(hydroxymethyl)aminomethane buffer (pH 8.4) containing 2 mm MgCl(2)]. The purified alkaline phosphatase also acted on all these esters, although much less effectively on adenosine triphosphate and sodium pyrophosphate than did the cells. Comparison of the relative utilization of the various substrates by repressed and derepressed cells and purified enzyme suggested the presence of multiple enzymes in the cells. Thus, the cytochemical method of trapping the newly generated inorganic phosphorus determines the location of an alkaline phosphatase of broad substrate profile, and in addition locates the sites of other enzymes generating inorganic phosphorus under identical conditions of assay. It is intriguing that all of these enzymes usually exist in a few clusters attached to the peripheral plasma membrane. In addition to this predominant location, there were a few sites of enzyme activity in the cytoplasm unattached to any discernible structure, and also in the cell wall of the repression-resistant and of the derepressed, repressible strains.     

Modulation of receptor-mediated signal transduction by diacylglycerol mimetics in astrocytes

1. When rat astrocytes in primary culture were incubated with bradykinin, inositol phosphate formation and arachidonic acid release were stimulated. 2. By themselves, phorbol esters inhibited inositol phosphate formation, but phorbol esters and other cell-permeant diacylglycerol analogues stimulated arachidonic acid release. Preincubation of the cells with phorbol esters or diacylglycerol analogues blocked bradykinin-stimulated inositol phosphate formation but augmented bradykinin-stimulated arachidonic acid release. 3. The present results suggest that, in astrocytes, bradykinin activates at least two signal transduction pathways bradykinin stimulates a phosphatidylinositol-specific phospholipase C leading to enhanced inositol phosphate formation, and bradykinin stimulates a second phospholipase to enhance arachidonic acid release. The pathways may be distinguished using phorbol esters and other diacylglycerol mimetics. 4. The possibility is raised that diacylglycerol, formed in response to bradykinin, may serve as a transducer of receptor-receptor interactions by altering the ability of receptors to stimulate phospholipase activity.     

Molecular and catalytic properties of a butyrylesterase from human red cells and brain

A butyrylesterase from human red cells was prepared to homogeneity using DEAE-cellulose, Ultrogel ACA-34, DEAE-Sephacel, and precipitation with 1.5 M (NH4)2SO4. The yield was 25-35% relative to the enzyme activity of the hemolysate. Because of its preference for butyric acid esters the enzyme was designated a butyrylesterase. With alpha-naphthyl butyrate the Km was 7.6 microM and the kcat, 48 s-1. The molecular weight was 340,000 and the subunit weight 85,000, indicating a tetrameric structure. The isoelectric pH was 4.0. The enzyme preparation did not contain cystine. Sialic acid or other carbohydrate components could not be detected. The enzyme was irreversibly inhibited by organophosphate esters and the second-order rate constant was 192 M-1 s-1 for diethyl p-nitrophenyl phosphate. For the brain enzyme the constant was 206 M-1 s-1. The enzyme was irreversibly inhibited by sulfhydryl reagents, indicating that the enzyme is a sulfhydryl-dependent serine esterase. The enzyme was identical to the butyrylesterase from human brain, and the two enzymes were immunochemically identical. An amino acid ester has been shown to be split at a higher rate than butyric acid esters; however, the specificity constant (kcat/Km) was lower for the amino acid ester than for the butyric acid ester. The enzyme did not exhibit amidase activity.     

Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex.

Two forms of tartrate-sensitive acid phosphatases (EC 3.1.3.2) were purified from rabbit kidney cortex by a multiple-column-chromatography method. The basic form constituted 90% of the enzyme and migrated as a single band of protein on polyacrylamide-gel electrophoresis. The proteins contaminating the acidic form did not exceed 5% of the total protein. The specific activity towards p-nitrophenyl phosphate was 12 mumol/min per mg for the basic form and 0.7 mumol/min per mg for the acidic form. The basic form of the enzyme differs from the acidic form in its heat-stability, Km values, inhibition rates by tartrate and fluoride and substrate specificities. Relative to p-nitrophenyl phosphate hydrolysis rate, the acidic form hydrolysed a variety of physiological monophosphate esters, whereas the basic form hydrolysed only CMP and phosphoenolpyruvate. Bacterial neuraminidases had no effect on the activity and mobility of the acidic form on polyacrylamide-gel electrophoresis. Both forms have the same molecular weight (101000 +/- 4000) and are probably composed of two identical subunits. The question whether the two forms of the enzyme are different proteins or whether one is a modified form of the other is discussed.     

Characterization of an inducible amidase from Pseudomonas acidovorans AE1.

The main molecular and catalytic properties of an acetanilide-hydrolyzing enzyme from Pseudomonas acidovorans AE 1, purified to a homogeneous state, were investigated. The molecular weight was 57 500 as determined by gel filtration and 55 300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56 700 was obtained. Based on the reaction of the highly purufied enzyme with diethyl-4-nitrophenyl phosphate an equivalent weight of approximately 59 100 was found. From these results it was concluded that the enzyme consists of a single polypeptide chain and contains one active site per molecule. The enzyme hydrolyzed esters as well as certain aromatic amides. It also catalysed the transfer of acetyl groups to phenetidine yielding phenacetin. The activities towards aliphatic esters were much smaller. The enzyme was stable at pH values ranging from 7 to 9 and its pH-optimum was about 10. It was strongly inhibited by organophosphorous compounds, like diethyl-4-nitrophenyl phosphate or diisopropylphosphorofluoridate, as well as by physostigmine sulfate and -SH-blocking reagents, like HgCl-2 or 4-chloromercuribenzoic acid. o-Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition.     

Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates

Kinetic properties of rat liver acid phosphatase were evaluated using the conventional synthetic substrates sodium beta glycerophosphate (betaGP) and p-nitrophenyl phosphate (PNPP) and physiologically occurring phosphate esters of carbohydrates, vitamins and nucleotides. The extent of hydrolysis varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kinetic analysis revealed the presence of two components of the enzyme for all the substrates. Component I had low Km and low Vmas. Opposite was true for component II. The Km values were generally high for betaGP, PNPP and adenosine diphosphate (ADP). Amongst the nucleotides substrates AMP showed high affinity i.e. low Km. The increase in enzyme activity in general at high substrate concentration seems to be due to substrate binding and positive cooperativity. AMP which showed highest affinity was inhibitory at high concentration beyond 1 mM. The results suggest that in situ the nucleotides may be the preferred substrates for acid phosphatase.     

Isolation of D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta

We have isolated D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase (EC 3.1.4.36) from human placenta. This enzyme catalyzes the conversion of inositol 1:2-cyclic phosphate to inositol 1-phosphate. The enzyme was purified 1300-fold to apparent homogeneity from the soluble fraction of human placenta. The enzyme requires Mn2+ or Mg2+ ions for activity, has an apparent Km for inositol 1:2-cyclic phosphate of 0.15 mM and forms 2.2 mumol of inositol 1-phosphate/min/mg protein. The enzyme does not utilize the cyclic esters of inositol polyphosphates as substrates. The molecular weight determined by gel filtration chromatography is approximately 55,000. Upon electrophoresis in polyacrylamide gels in sodium dodecyl sulfate, the molecular weight was found to be 29,000 both in the presence and absence of beta-mercaptoethanol. The enzyme was inhibited by inositol 2-phosphate (IC50 = 4 microM) and to a lesser degree by inositol 1-phosphate (IC50 = 2 mM) and inositol (IC50 = 4 mM). Zn2+ is a potent inhibitor of enzyme activity (IC50 = 10 microM). Neither Li+ nor Ca2+ had any effect on enzyme activity. This enzyme may serve to generate inositol from inositol cyclic phosphate metabolites produced by the phosphoinositide signaling pathway in cells.     

Purification and properties of phytate-specific phosphatase from Bacillus subtilis.

An enzyme which liberates Pi from myo-inositol hexaphosphate (phytic acid) was shown to be present in culture filtrates of Bacillus subtilis. It was purified until it was homogeneous by ultracentrifugation, but it still showed two isozymes on polyacrylamide gel electrophoresis. The enzyme differed from other previously known phytases in its metal requirement and in its specificity for phytate. It had a specific requirement for Ca2+ for its activity. The enzyme hydrolyzed only phytate and had no action on other phosphate esters tested. This B. subtilis phytase is the only known phytate-specific phosphatase. The products of hydrolysis of phytate by this enzyme were Pi and myo-inositol monophosphate. The enzyme showed optimum activity at pH 7.5. It was inhibited by Ba2+, Sr2+, Hg2+, Cd2+, and borate. Its activity was unaffected by urea, diisopropylfluorophosphate, arsenate, fluoride, mercaptoethanol, trypsin, papain, and elastase.     

Nucleoside phosphotransferase from Erwinia herbicola, a new membrane-bound enzyme.

A nucleoside phosphotransferase, which catalyzes the phosphorylation of nucleosides to nucleotides by low energy phosphate esters, has been isolated and purified 500-fold from the membrane fraction of Erwinia herbicola. Its most noteworthy difference from other enzymes of this class is that it is membrane bound and can be isolated and handled only in the presence of a detergent. With a ribonucleoside acceptor, adenosine, the reaction product is exclusively 5'-AMP; with deoxyadenosine, 5'- and 3'-nucleotide products appear in the approximate ratio of 2:1, respectively. The enzyme has no detectable phosphatase activity with the best phosphate donors, 5'-dAMP and 5'-dTMP, and very little with less active donors, such as p-nitrophenyl phosphate. This phosphotransferase should be a useful agent for preparing 5'-nucleotides from unusual synthetic bases.     

Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.     

Purification and properties of inorganic pyrophosphatase from porcine brain

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from porcine brain to an electrophoretically homogeneous state. The molecular weight of the enzyme was estimated to be 62,000 by gel filtration and that of the subunit to be 33,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme consists of two identical subunits. The stability of the purified enzyme was dependent on its protein concentration. The enzyme was stable above 50 micrograms/ml at 20 degrees C, but it was gradually inactivated below this concentration, even at 0 degree C unless other proteins such as bovine serum albumin, calmodulin, etc. were present. Those added proteins not only protected the enzyme from inactivation, but also completely reactivated the enzyme after it had been once inactivated. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate but not that of other phosphate esters. Only Mg2+ was required as an activating cation, and other divalent cations inhibited the activity to some degree. The addition of sulfhydryl reagents prevented the inhibition of activity by divalent cations.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.     

Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits.     

Biosynthesis of monoterpenes: hydrolysis of bornyl pyrophosphate, an essential step in camphor biosynthesis, and hydrolysis of geranyl pyrophosphate, the acyclic precursor of camphor, by enzymes from sage (Salvia officinalis).

Soluble enzyme preparations from sage (Salvia officinalis) leaves catalyze the hydrolysis of (+)-bornyl pyrophosphate to (+)-borneol, which is an essential step in the biosynthesis of the cyclic monoterpene (+)-camphor [(1R,4R)-bornan-2-one] in this tissue. Chromatography of the preparation on Sephadex G-150 allowed the separation of two regions of bornyl pyrophosphate hydrolase activity. One region was further separated into a pyrophosphate hydrolase and a monophosphate hydrolase by chromatography on hydroxylapatite, but the other contained pyrophosphate and monophosphate hydrolase activities which were inseparable by this or any other chromatographic technique tested. Each phosphatase and pyrophosphatase activity was characterized with respect to molecular weight, pH optimum, response to inhibitors, K_m for bornyl phosphate or bornyl pyrophosphate, and substrate specificity, and each activity was distinctly different with regard to these properties. One pyrophosphatase activity was specific for pyrophosphate esters of sterically hindered monoterpenols such as bornyl pyrophosphate. The other preferred pyrophosphate esters of primary allylic alcohols such as geranyl pyrophosphate and neryl pyrophosphate, which are precursors of cyclic monoterpenes, and it hydrolyzed geranyl pyrophosphate at faster rates than neryl pyrophosphate. The monophosphate hydrolase activities were similar in substrate specificity, showing a preference for phosphate esters of primary allylic alcohols. The terpenyl pyrophosphate hydrolase exhibiting specificity for bornyl pyrophosphate may be involved in camphor biosynthesis in vivo, while the terpenyl pyrophosphate hydrolase more specific for geranyl pyrophosphate was shown to be a source of potential interference in studies on monoterpene cyclization processes.     

Further characterization of mitochondrial protein phosphatase.

Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate.Aliphatic phosphate esters (β-glycerophosphate, glucose-6-P, serine-phosphate) are not dephosphorylated to any detectable extent.Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in vivo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.     

Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans.

An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.