Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.     

Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul

1. Partially purified ceramide trihexoside alpha-galactosidase from human liver was studied by using ceramide trihexoside specifically tritiated in the terminal galactose. 2. The hydrolysis of ceramide trihexoside was absolutely dependent on a mixture of sodium taurocholate and Triton X-100 and was markedly inhibited by human serum albumin and by NaCl. 3. The Lineweaver-Burk plot for ceramide trihexoside hydrolysis was upward curving. Ceramide lactoside inhibited hydrolysis of all concentrations of ceramide trihexoside. Ceramide digalactoside stimulated hydrolysis of low concentrations of ceramide trihexoside, but inhibited hydrolysis of high concentrations of the lipid. 4. alpha-Galactosidase activity assayed with the synthetic substrate 4-methylumbelliferyl alpha-d-galactopyranoside fractionated together with activity assayed with the natural substrate ceramide trihexoside. Both activities had identical heat-inactivation kinetics. 5. Characteristics of the hydrolysis of the synthetic substrate differed considerably from those of the natural substrate, including pH optimum, shape of the Lineweaver-Burk plot, and differential effects of inhibitors and activators. Mutual inhibition of hydrolysis between the synthetic and natural substrates was predominantly non-competitive. 6. These results are discussed in the light of special problems involved in the hydrolysis of lipids in an aqueous milieu.     

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).     

Evidence for a phosphoenzyme intermediate in the reaction pathway of rat hepatic fructose-2,6-bisphosphatase

We re-examined the kinetics of the bisphosphatase reaction of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase after depleting the enzyme of bound fructose 6-phosphate and found a hyperbolic dependence on fructose 2,6-bisphosphate at concentrations below 100 nM. The Michaelis constant was 4 nM, the Vmax was about 12 nmol X mg-1 X min-1 at 22 degrees C but the substrate inhibited at concentrations above 100 nM. Both phosphate and alpha-glycerol phosphate strongly inhibited phosphoenzyme formation and hydrolytic rate below 100 nM, but relieved the inhibition by substrate at higher concentrations probably by antagonizing substrate binding. A number of observations support the proposition that the phosphoenzyme is a necessary participant in catalysis. 1) The amount of phosphoenzyme measured during steady-state hydrolysis as a function of substrate concentration correlated with the velocity profile. 2) Rapid mixing experiments demonstrated that over a broad range of substrate concentrations phosphoenzyme formation was faster than the net rate of hydrolysis. 3) Both phosphate and alpha-glycerol phosphate inhibited the rate of phosphoenzyme formation and, at low substrate concentrations, reduced the steady-state phosphoenzyme levels. The latter correlated with inhibition of substrate hydrolysis. 4) Both phosphate and alpha-glycerol phosphate stimulate the rate of phosphoenzyme breakdown, consistent with their stimulation of substrate hydrolysis at high substrate concentrations. 5) The fractional rate of phosphoenzyme breakdown, which was pH and substrate dependent, multiplied by the amount of phosphoenzyme obtained in the steady state at that pH and substrate concentration approximated the observed rate of hydrolysis. We conclude that the phosphoenzyme is a reaction intermediate in the hepatic fructose-2,6-bisphosphatase reaction.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Biosynthesis of phosphatidylinositol in Crithidia fasciculata

Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.     

Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase.

Sphingomyelin in mixed dispersion with bile salts was hydrolysed by the solubilized sphingomyelinase of rat brain lysosomes. In parallel studies, physical properties of these dispersions were determined. The kinetic curves that described the rate of hydrolysis as a function of increasing concentrations of bile salt were multiphasic. A region of very low activity was followed by an ascending portion, a peak, a descending portion, a trough and a second ascending portion. The positions of the initiation points, peaks and troughs were found to be a function of the respective ratios of the bile salt to sphingomyelin for the detergent sodium taurodeoxycholate, but of the absolute concentration of the detergent for sodium taurocholate. Turbidity studies suggested that hydrolysis of sphingomyelin begins at a bile salt concentration that solubilizes the lipid and incorporates it into a mixed micelle with the detergent. Ultracentrifugation studies suggested that the sizes of the mixed aggregates of detergent and lipid were a function of the ratio of taurodeoxycholate to sphingomyelin, but of the absolute concentration of the bile salt, for sodium taurocholate.     

Activity of human hepatic beta-galactosidase toward natural glycosphingolipid substrates.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.     

The effect of detergents on immunoprecipitability of lysosomal sphingomyelinase

Antibodies raised against the soluble form of acid sphingomyelinase from human urine and placenta are able to precipitate about 70% of the sphingomyelinase activity present in preparations of urinary sphingomyelinase. In contrast, no precipitation of sphingomyelinase activity occurs in detergent-containing preparations from placenta or splenic membranes. The formation of immune complexes between the antibodies and urinary sphingomyelinase is inhibited if detergents are added. With the non-ionic detergent Triton X-100 significant inhibition occurs only above the critical micellar concentration of the detergent. With the anionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (Chaps) substantial inhibition is already observed below the critical micellar concentration of the detergent.     

Magnesium-dependent sphingomyelinase of infantile brain. Effect of detergents and a heat-stable factor.

The properties of the Mg2+-dependent sphingomyelinase, whose pH optimum is between 7 and 8, were investigated using post-mortem infantile brain. The enzyme could be extracted with 0.2% Triton X-100 and remained soluble when centrifuged at 170,000 X g. Subsequent removal of the detergent with SM2-Biobeads resulted in resedimentation of the enzyme at 80,000 X g. A detergent was needed for assaying enzymatic activity; either Triton X-100 or bile salts could be used. With increasing concentrations of detergent, the rates of hydrolysis of sphinomyelin increased, reached an optimum and then decreased, suggesting inhibition of the enzyme. The concentrations of detergent which resulted in optimal reaction rates were directly related to the protein concentration of the enzymatic preparation. A heat-stable factor which counteracts inhibition by the above detergents is present in brain as well as several other tissues. A lipid extract of the enzymatic preparation, or several purified lipids could not mimic the effect of the heat-stable factor. The interrelationship between enzyme, detergent and the heat-stable factor was investigated.     

Inhibition or activation of Pseudomonas species lipase by 1,2-ethylene-di-N-alkylcarbamates in detergents

1,2-Ethylene-di-N-n-propylcarbamate (1) is characterized as an essential activator of Pseudomonas species lipase while 1,2-ethylene-di-N-n-butyl-, t-butyl-, n-heptyl-, and n-octyl-carbamates (2-5) are characterized as the pseudo substrate inhibitors of the enzyme in the presence of the detergent taurocholate or triton X-100. The inhibition and activation reactions are more sensitive in taurocholate than in triton X-100. From CD studies, the enzyme changes conformations in the presence of the detergent and further alters conformations by addition of the carbamate activator or inhibitor into the enzyme-detergent adduct. Therefore, this study suggests that the conformational change of lipase during interfacial activation is a continuous process to expose the active site of the enzyme to substrate. From 600 MHz (1)H NMR studies, the conformations of the alpha- and beta-methylene moieties of the activator 1,2-ethylene-di-N-n-propylcarbamate in the presence of substrate change after adding taurocholate into the mixture, and the conformations of the beta-methylene moieties of the inhibitor 1,2-ethylene-di-N-n-butylcarbamate in the presence of substrate alter after adding taurocholate into the mixture.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.     

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates.

The effects of Ca2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca2+ and Mg2+.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

Physicochemical and physiological properties of 5alpha-cyprinol sulfate,the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.

Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.     

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.