Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

he sulphatase of ox liver XVII. Sulphatase A as a glycoprotein

The glycoprotein nature of sulphatase A has been confirmed. The monomer of sulphatase A (mol. wt 107 000) contains eight molecules of glactose, 14 of mannose, 18 of glucosamine and eight of sialic acid together with traces of focuse and glucose. The latter may be contaminant. Treatment of sulphatase A with neuraminidase quantitatively removes the sialic acid showing that this must be in the terminal position of the carbohydrate. The desialylated enzyme retains the properties of native sulphatase A apart from a slight change in charge and it is quite distinct from sulphatase B. The desialylated enzyme still hydrolyses cerebroside sulphate.The implications of these findings in the biochenmistry of metachromatic leucodystrophy are considered.     

Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.     

Bile salt activation of cerebroside sulphate sulphohydrolase.

The cholate and taurodeoxycholate activations of cerebroside sulphate sulphohydrolase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) activity of arylsulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) were compared. Taurodeoxycholate caused a sharp peak of response at a concentration of 1.25 mg/ml (type-I activation). Cholate also showed type-I activation but, in addition, it evoked a second, higher, response plateau at concentrations between 5 and 10 mg/ml (type-II activation). At the pH of the reaction, cholate is converted largely to the sparingly soluble free aicd, so at the high concentrations associated with type-II activation, copious precipitates were formed. It was found that the precipitated material was essential for the type-II activation. Type-I activation appears to involve bile salt interaction with substrate, while type-II activation appears to involve enzyme interaction with solid-phase cholic acid. the putative mutant arylsulphatase A in an unusual form of metachromatic leukodystrophy hydolysed cerebroside sulphate only in the presence of high levels of cholate. The type-II activation may thus be simulating a physiological desulphation reaction.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

CHOLESTEROL ESTER METABOLIZING ENZYMES IN HUMAN BRAIN: PROPERTIES, SUBCELLULAR DISTRIBUTION AND RELATIVE LEVELS IN VARIOUS DISEASED CONDITIONS

We have in the present study examined the properties and subcellular distribution of cholesterol ester metabolizing enzymes in human brain, and compared the levels of these enzymes in brains from patients with phenylketonuria (PKU), metachromatic leucodystrophy (MLD), and Down's Syndrome (DS). Cholesterol esterification was optimal at pH 5.6, did not require ATP or CoA as cofactors and was inhibited by detergents (TWEEN-20 and Triton X-100) and bile acids (sodium taurocholate and sodium deoxycholate). The specific activity of the cholesterol esterifying enzyme was highest in the mitochondrial fraction. Cholesterol esterifying activity in brains from PKU, MLD, and DS patients was not significantly different. Cholesterol ester hydrolase activity in human brain peaked at two different pHs (4.5 and 6.5). The activity was optimal when the substrate was dispersed in Triton X-100 and sonicated. The specific activity of the pH 4.5 hydrolase was highest in the mitochondrial fraction, while that of the pH 6.5 hydrolase was highest in myelin. The sulfhydryl group reagent parachloromercuribenzoate (PCMB) inhibited the activity of the hydrolase(s) but diisopropylfluorophosphate (DFP), a typical serine reagent, had no effect on hydrolase(s) activity. Addition of either phosphatidyl serine or phosphatidyl inositol significantly enhanced the hydrolase activity at both pHs. The level of cholesterol ester hydrolase(s) in PKU brains was lower than in the brains from DS patients, and the level of these enzymes in the brains from two patients with metachromatic leucodystrophy was lower than in the brains from PKU patients. It is concluded that the properties and subcellular distribution of cholesterol esterifying enzyme in human brain is similar to that in rat brain (Ero & Suzuki , 1971) but that the hydrolases in human brain differ from that in rat brain in several respects, and that the low levels of hydrolase(s) activity in MLD and PKU brain may be related to reduced myelin content of those brains.     

THE PATTERNS OF ARYLSULPHATASES A AND B IN HUMAN NORMAL AND METACHROMATIC LEUCODYSTROPHY TISSUES AND THEIR RELATIONSHIP TO THE CEREBROSIDE SULPHATASE ACTIVITY

Abstract— The arylsulphatase A and B patterns of human tissues and leucocytes have been established by isoelectric focussing. Assay conditions, which enable an evaluation of these patterns as quantitatively as possible, have been studied. The dependences of the enzyme patterns on the origin of the tissues and on the storage conditions have been determined. The arylsulphatase A obtained by isoelectric focussing exhibits cerebroside sulphatase activity in the presence of detergents. A purified preparation of the arylsulphatase B likewise shows a significant, although low, cerebroside sulphatase activity. In cases of the conventional types of metachromatic leucodystrophy the arylsulphatase A activity is missing, while in an atypical form of this disease ('ML Variant' according to A ustin et al . (1965) the arylsulphatase A, B and C activities are deficient. In both forms, however, residual activities of the deficient enzymes could be detected which showed isoelectric points identical to those of the normal enzymes.
The following nomenclature is proposed: 'Variant B' for the conventional type, in which the arylsulphatase B activity is present, and 'Variant O' for the exceptional cases, in which all arylsulphatase activities are deficient. The significance of the cerebroside sulphatase activity of arylsulphatase B for a possible residual turnover of cerebroside sulphates in the conventional type of the disease is discussed.     

Biochemical,psychometric, and neuropsychological studies in heterozygotes for various lipidoses

Summary A sample of 38 clinically unaffected carriers for various lipidoses and their noncarrier relatives was studied with biochemical, psychological, and neuropsychological tests under blind conditions. The largest group of carriers was that for metachromatic leucodystrophy (MLD). The mean activity of arylsulphatase A or cerebroside sulphatase in the obligate carriers was 25%–30% of the control values, some heterozygotes showing little more activity than MLD patients. It was found that compared with the controls all heterozygotes (both obligate and facultative) differ unfavourably in some personality traits and in WISA subtests, including capacity for spatial cognition. These differences are especially obvious in a group of seven MLD carriers from the same family.With respect to reaction times, performance was significantly slower in MLD carriers, and particularly in those with enzyme activity lower than 30% of the control values.     

A study of various properties of beta-galactocerebrosidase from human chorion using synthetic fluorescent substrates

The properties of beta-galactocerebrosidase from human chorionic villi, cultured chorionic villi and cultured skin fibroblasts were compared, using 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGaL) as substrate. The effects of bile salt and Triton X-100 on beta-galactocerebrosidase were examined. It was shown that optimization of the HMGaL assay system requires the presence of pure sodium taurocholate and Triton X-100 at concentrations of 4.5 mM and 0.28 mM, respectively. The optimal pH value was found to be equal to 4.5-5.0; Km for the substrate was 0.03 mM. A comparison of beta-galactocerebrosidase from chorionic villi and cultured chorionic villi with the enzyme from skin fibroblasts revealed the similarity of some properties of these enzymes. The experimental results suggest that HMGaL can be used as a substrate for the identification of chorionic villi beta-galactocerebrosidase in an early prenatal diagnosis of Krabbe's disease.     

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.     

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Inactivation of the calcium-transport ATPase in the sarcoplasmic reticulum by the combined effect of lasolocid and Triton X-100

The calcium-transport ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately, 0.2 mg X ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescence intensity of Lasolocid passes a distinct maximum at the most effective Triton X-100 concentration of 0.2 mg X ml-1.     

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.     

Substrate specificity of human alpha-L-fucosidase.

Human α-l-fucosidase is a soluble lysosomal enzyme which hydrolyzes α-l-fucose residues linked to the 2 position of galactose or the 3, 4, or 6 position ofN-acetylglucosamine. Demonstration of activity towards natural oligosaccharide or glycosphingolipid substrates was achieved by measuring liberated l-fucose by coupling to fucose dehydrogenase and NAD and measuring NADH production spectrophotometrically. Activity of purified human spleen, brain, and cultured skin fibroblast or crude cell extracts towards 4-methylumbelliferyl-α-l-fucoside had a pH optimum of 4.5 to 5.5 and was unaffected by the presence of neutral detergents such as Triton X-100. However, the addition of sodium taurocholate or other bile salts to the incubation mixture caused a marked inhibition at pH 5 and a shift in pH optimum to the pH 6–7 region. Sodium taurocholate effected a threefold reduction in the apparent K_m for α-l-fucosidase at pH 6.0, but studies on fucosidosis tissue (α-fucosidase deficiency) or subcellular fractions derived from rat liver failed to indicate the existence of a membrane-bound α-l-fucosidase. The response of other lysosomal hydrolases to the presence of bile salts was investigated and was found to be variable, perhaps depending upon the hydrophilic or hydrophobic nature of the natural substrate and/or the state of association of the active enzyme.     

Detergent activation and solubilization of 2'':3''-cyclic nucleotide 3''-phosphodiesterase from isolated myelin and c6 cells.

Several detergents were investigated for their ability to increase activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. The ability of Triton X-100 and Sulfobetaine DLH to solubilize the enzyme was also examined. Solubilization with Triton X-100 was only effective in the presence of salt, for example with NaCl 51% of the activity was solubilized. A single extraction with Sulfobetaine DLH yielded slightly more solubilized enzyme and did not require added salt. Both activation and solubilization of 2':3'-cyclic nucleotide 3'-phosphodiesterase appeared to be similarly dependent on detergent concentration, suggesting a common action of the detergent in the two processes. Myelin basic protein was solubilized more readily than the enzyme. In contrast with the enzyme in myelin, 2':3'-cyclic nucleotide 3'-phosphodiesterase activity in C6 cells was not increased in the presence of Triton X-100, and was partially solubilized by either Triton X-100 or NaCl alone. No myelin basic protein could be detected in C6 cells by radioimmunoassay.     

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.     

Evidence for the Presence of CDP-Ethanolamine: 1,2-diacyl-sn-glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin

Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Solubilization and stabilization of human liver glycoprotein sialyltransferase.

Triton X-100 is increasingly effective in solubilizing human liver glycoprotein (asialofetuin) sialytransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotien N-acetylneuraminyltransferase, EC 2.4.99.1) activity as its concentration is increased in the homogenizing buffer. At the optimal concentration of 1.5% (v/v), essentially all of the homogenate sialyltransferase activity was solubilized into the supernatant fluid. Higher concentrations of Triton X-100 inhibited sialyltransferase activity. Several kinetic properties of the solubilized asialofetuin-sialyltransferase activity were compared to those of the membrane-bound enzyme(s) (in homogenates made without Triton X-100 or in resuspended pellets). No major difference was apparent, suggesting that solubilization has not significantly altered the properties of sialyltransferase. The solubilized sialyltransferase activity is quite unstable, losing approximately 50% of its activity after one week of storage at 4 degrees C. Various detergents (Zwittergent, sodium taurocholate and sodium deoxycholate) are differentially effective in stabilizing the solubilized activity. Sodium taurocholate (1.5%, w/v) was most effective with no loss in activity after 40 days and minimal loss (14%) after 60 days storage at 4 degrees C. The solubilized sialyltransferase preparation retains full activity after storage in the frozen state (-20 degrees C) for at least 159 days.