A.S. Balasubramanian

A CONTROLLED STUDY OF ENZYMIC ACTIVITIES IN THREE HUMAN DISORDERS OF GLYCOLIPID METABOLISM.

A CONTROLLED STUDY OF ENZYMIC ACTIVlTIES IN THREE HUMAN DISORDERS OF GLYCOLIPID METABOLISM” JAMES H. AUSTIN Division of Neurology, University of Oregon Medical School, Portland, Oregon, U.S.A. A . s. BALASUBRAMANIAN~, T. N. PATTABIRAMAN

The distribution of estrone sulphatase, dehydroepiandrosterone sulphatase, and arylsulphatase C in the primate (Macaca radiata) brain and pituitary

, s. SARASWATHI, D. K. BASUS and B. K. BACHHAWAT Neurochemistry Laboratory, Department of Neurology and Neurosurgery, Christian Medical College & Hospital, Vellore, S. India

Serotonin-sensitive aryl acylamidase activity of platelet acetylcholinesterase

Abstract: Estrone sulphatase, arylsulphatase C and dehydroepiandrosterone sulphatase were measured in the pituitary, hypothalamus‐preoptic area, amygdala, hippocampus, midbrain, septum, frontal cortex and occipital cortex of monkey (Macaca radiata) brain. All the regions showed measurable activities of all three enzymes. In all the animals tested, either the midbrain or hypothalamus‐preoptic area showed the greatest activity of all three enzymes. In particular, estrone sulphatase showed the highest specific activity in either of the above two regions in all animals. Subcellular distribution studies in the hypothalamus preoptic area showed a similarity in the distribution profile between arylsulphatase C and estrone sulphatase and a significant difference of dehydroepiandrosterone sulphatase from the others.

The solubilization of platelet membrane-bound acetylcholinesterase and aryl acylamidase by exogenous or endogenous phosphatidylinositol specific phospholipase C

Serotonin-sensitive aryl acylamidase (AAA, EC 3.5.1.13) was purified to apparent homogeneity from sheep platelets by affinity chromatography and it was shown to be associated with the platelet acetylcholinesterase (AChE, EC 3.1.1.7). The basis for the association of the two enzymes was the following. Both enzyme activities co-eluted from the affinity columns with constant ratios of specific activities and percentage recoveries. Both enzymes co-migrated on gel electrophoresis. Both enzymes co-eluted during sepharose 6B gel filtration. Potent inhibitors of AChE such as bis(4-allyldimethyl ammoniumphenyl) pentan-3-one dibromide (BW 284C51), neostigmine and eserine also inhibited AAA potently. Both enzymes lost significant activity on treatment with deoxycholate or taurodeoxycholate and the loss could be partly restored by a mixture of phospholipids. The platelet AAA was specifically inhibited by serotonin and to a lesser extent by tryptamine but not by several other amines. It was also inhibited by acetylcholine and several of its analogues and homologues. It is suggested that in the platelets the two enzymes (AAA and AChE) are probably identical.

Arylsulphatase C and Estrone Sulphatase of Sheep Hypothalamus, Preoptic Area, and Midbrain: Separation by Hydrophobic Interaction Chromatography and Evidence for Differences in Their Lipid Environment

Phosphatidylinositol specific phospholipase C from Staphylococcus aureus could solubilize acetylcholinesterase up to 55% from sheep platelets in the presence of ethylenediaminetetra acetic acid (EDTA). The endogenous phosphatidylinositol specific phospholipase C of platelets activated by deoxycholate (at 3-5 mM) could also solubilize the enzyme to a similar extent. The solubilized enzyme could be further purified to apparent homogeneity by affinity chromatography without the use of any detergents. It is suggested that phosphatidylinositol specific phospholipase C will be a useful tool in the solubilization of acetylcholinesterase from mammalian sources and its purification free of detergents. The present study also demonstrates the parallel behaviour of acetylcholinesterase and aryl acylamidase in platelets confirming their identity.

The inhibition of brain aryl acylamidase by 5-hydroxytryptamine and acetylcholine

Abstract: Arylsulphatase C and estrone sulphatase activities of sheep hypothalamus‐preoptic area‐midbrain were examined for their susceptibility to phospholipase action. Russels viper phospholipase A could completely inactivate estrone sulphatase without affecting arylsulphatase C. The latter was partially inactivated by S. aureus phospholipase C but not by C. welchi phospholipase C. Both arylsulphatase C and estrone sulphatase were inactivated to different extents by sodium deoxycholate, which is known to activate the intrinsic phospholipases of brain. Hydrophobic interaction chromatography on phenyl‐Sepharose resulted in the differential elution of arylsulphatase C and estrone sulphatase. The results suggest that one enzyme is not responsible for arylsulphatase C and estrone sulphatase activities.

Lysosomal and microsomal β-glucuronidase of monkey brain: Differential elution characteristics from con a-sepharose and neutral sugar composition

Abstract Sheep brain aryl acylamidase (aryl-acylamide amidohydrolase. EC 3.5.1.13) was partially purified. Of a number of amines tested at 1 mM the enzyme was maximally inhibited by 5-hydroxytryp-tamine (5-HT) and to a lesser extent by tryptamine and practically unaffected by tyramine, histamine, noradrenaline, dopamine and benzylamine and a number of amino acids including tryptophan. Choline derivatives at 1 mM were inhibitory to the enzyme in the order of butyrylcholine >succinylcholine > benzoylcholine > choline > acetylcholine > acetylthiocholine > acetyl-β-methylcholine > pro-pionylcholine. The inhibition by 5-HT and acetylcholine was further studied. Both inhibit the enzyme in a noncompetitive manner. The inhibition could be reversed by removal of the inhibitors from the enzyme by gel filteration. A number of metal ions, EDTA, high concentrations of sodium chloride. the thiol reagents p -chloromercuribenzoate, N -ethyl-maleimide and iodoacetamide were found to have no effect on the inhibition. Dithiothereitol as well as neuraminidase treatment did not alter the extent of inhibition of the enzyme by 5-HT or acetylcholine. Nitration of the enzyme with tetranitromethane led to approximately a 50 per cent drop in enzyme activity as well as a significant decrease in the extent of inhibition by 5-HT and acetylcholine. This suggested the possibility of the involvement of tyrosine residues both at the catalytic site as well as at the site(s) of inhibition by 5-HT and acetylcholine. Mixed inhibitor studies favoured a common inhibition site for both 5-HT and acetylcholine on the brain enzyme. The sheep liver enzyme was not inhibited by either 5-HT or acetylcholine.

Aryl acylamidase of monkey brain and liver: Response to inhibitors and relationship to acetylcholinesterase

Microsomal and lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) of monkey brain were differentially eluted from Con A-Sepharose when subjected to chromatography and linear gradient elution with methyl alpha-glucoside at 28+/-1 degree C. The lysosomal enzyme was eluted as a sharp peak in the first few fractions, while the microsomal enzyme was eluted as a broad peak extending over several fractions. This differential pattern of elution was dependent only on the temperature of elution and the concentration of methyl alpha-glucoside used. The lysosomal and microsomal glucuronidases were purified to apparent homogeneity and their neutral sugar analysed. Both of them contained glucose, mannose and fucose but the microsomal enzyme contained about 3-times as much of all these sugars as the lysosomal enzyme. Sodium periodate treatment of the microsomal enzyme resulted in a shift in its elution pattern, similar to the lysosomal enzyme when subjected to Con A-Sepharose chromatography. The content of neutral sugars and the structural features of the oligosaccharide units in the microsomal glucuronidase might be responsible for its elution pattern. A processing of the carbohydrate units of the microsomal glucuronidase might be envisaged to take place if it were to act as a precursor of the lysosomal glucuronidase.

The peptidase activity of human serum butyrylcholinesterase : studies using monoclonal antibodies and characterization of the peptidase

Abstract The serotonin sensitive aryl acylamidase (aryl acylamide amidohydrolase EC 3.5.1.13) of monkey brain was compared with the liver enzyme. Although the two enzymes showed some similarities in their properties such as pH optima, the effect of metal ions and thiol agents, they significantly differed in their mol. wt and response to inhibitors. The liver enzyme had a higher mol. wt as observed by gel filtration on Sepharose 6B and a greater heat stability. The brain enzyme was inhibited specifically by the amines serotonin and tryptamine as well as by acetylcholine and its analogues and homologues in a non-competitive manner. The liver enzyme was unaffected by the above mentioned amines or acetylcholine but it was non-competitively inhibited by indole-3-acetic acid and indole-3-propionic acid both compounds having no effect on the brain enzyme. Eserine, a strong competitive inhibitor of acetylcholinesterase, at 10 −7 M inhibited the brain aryl acylamidase to 75 per cent leaving the liver enzyme unaffected. Eserine inhibition of the brain enzyme was non-competitive. From Dixon plots serotonin, acetylcholine and eserine were shown to act at the same site on brain aryl acylamidase. The inhibition of the brain enzyme by eserine and acetylcholine, the elution of both aryl acylamidase and acetylcholinesterase activities in the same fractions during gel filtration and the regional distribution of aryl acylamidase in the brain suggested the association of aryl acylamidase with acetylcholinesterase in the brain though not in the liver.

A binding protein for lysosomal enzymes isolated from brain by Phosphomannan-Sepharose chromatography

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited α-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.