Studies on the chaotropically solubilized arylsulfatase C and estrone sulfatase of sheep brain.

Arylsulfatase C (aryl-sulfate sulfohydrolase, EC 3.1.6.1) from sheep brain acetone powder was solubilized with the chaotropic agent, KSCN. Anti-chaotropes such as (NH4)2SO4 or sodium citrate significantly enhanced the activity of the solubilized enzyme indicating that hydrophobicity was an important factor influencing the enzyme activity. Dialysis or gel filtration of the solubilized enzyme resulted in a marked loss of activity. 3a dialyzable activator could reconstitute the activity in the presence of the antichaotropes. The activator was purified partially and preliminary studies indicated it to be a low molecular weight peptide. Arylsulfatase C and estrone sulfatase activities were compared in the solubilized enzyme. Estrone sulfatase activity was also increased in the presence of antichaotropes at lower concentration in comparison to arylsulfatase C. It however did not show a requirement for the dialyzable activator. Kinetic studies showed that elevation of enzyme activity by the antichaotropes and activator in the case of arylsulfatase C and by antichaotropes in the case of estrone sulfatase was due to an increase in V with a decrease in Km.

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.

Lysosomal and microsomal beta-glucuronidase of monkey brain. Differential elution characteristics from con A-sepharose and neutral sugar composition.

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.

Soluble arylsulfatases of human brain and some characteristics of the brain-specific arylsulfatase Bm.

The brain-specific arylsulfatase Bm (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was demonstrable in human and monkey brain. Arylsulfatases A, B and Bm were separated employing DEAE-cellulose chromatography. There was a distinct difference in the proportion of the sulfatases in infant and adult human brain. Arylsulfatase Bm after concanavalin A-Sepharose chromatography showed the property of binding to Sephadex G-200 totally. Several dissociating agents failed to elute the enzyme from the bound form. Under similar conditions arylsulfatase A did not show any binding to Sephadex. On treatment with Escherichia coli alkaline phosphatase adult human brain arylsulfatase Bm but not arylsulfatase A was converted into a less acidic, presumably dephosphorylated form that did not bind to DEAE-cellulose. Monkey brain arylsulfatase Bm showed a similar susceptibility to E. coli phosphatase treatment. Inorganic phosphate and serine phosphate but not mannose 6-phosphate could inhibit this dephosphorylation. There were differences in the susceptibilities to alkaline phosphatase treatment of the arylsulfatase Bm from infant and adult human brain. Endogenous phosphatase also seemed to have a role on the phosphorylated state of arylsulfatase Bm.

Stimulation by lysosomal enzymes and mannose-6-phosphate of a phosphoprotein phosphatase activity associated with the lysosomal enzyme binding receptor protein from monkey brain.

Previously we have isolated a lysosomal enzyme binding receptor protein from monkey brain that exhibits protein kinase activity and undergoes phosphorylation on serine and tyrosine residues. Using the 32P-labelled receptor protein, we have found that the lysosomal enzyme fucosidase and mannose-6-phosphate, which are ligands for the receptor, stimulated a protein phosphatase activity associated with the receptor protein. Stimulation of protein phosphatase activity using the 32P-labelled receptor protein was demonstrated both by the loss in radioactivity of the receptor and by the release of 32P-phosphate. There was no stimulation by a non-lysosomal glycoprotein enzyme, or by the sugars mannose or glucose. Both serine-phosphate and tyrosine-phosphate residues were dephosphorylated. Stimulation of protein phosphatase activity by fucosidase and mannose-6-phosphate was also demonstrated using as substrate histone 32P-labelled, on serine/threonine or tyrosine residues. Insulin-like growth factor II, another known ligand for the lysosomal enzyme binding receptor, did not show any significant effect, either on the phosphorylation or dephosphorylation of the receptor protein. Our previous and present results suggest that a phosphorylation/dephosphorylation mechanism may be operative in the ligand binding and functions of the receptor.

Selective inactivation of butyrylcholinesterase with metal chelators suggests there is more than one metal binding site.

Cholinesterases exhibit functions apart from their esterase activity. We have demonstrated an aryl acylamidase and a zinc stimulated metallocarboxypeptidase activity in human serum butyrylcholinesterase. To establish the presence of zinc binding sites in the enzyme we examined the effect of metal chelators on its catalytic activities. The metal chelators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)ethylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to 65Zn2+. Histidine modification of the enzyme pretreated with chelators resulted in abolition of 65Zn2+ binding and zinc-Sepharose binding. Whereas the binding studies demonstrated removal of a metal from a Zn2+ binding site, attempts to remove the metal responsible for catalytic activity were unsuccessful. Atomic absorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme after EDTA treatment. The results indicate that there are at least two metal binding sites on butyrycholinesterase. The presence of two HXXE...H sequences in butyrylcholinesterase supports these findings. Our studies implicate a zinc dependent metallocarboxypeptidase activity in the non-cholinergic functions of butyrylcholinesterase.

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

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.

Serotonin-sensitive aryl acylamidase activity of platelet acetylcholinesterase.

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.

Sulfation of proteins in the primate cerebellum and young rat brain.

Protein tyrosine sulfotransferase activity in a 20,000 g sedimentable fraction of monkey cerebellum was demonstrated. Both endogenous proteins and the exogenous substrate poly (Glu, Ala, Tyr) random copolymer were sulfated. The copolymer in the low molecular mass range (approx 20 kDa) was preferentially sulfated. Addition of copolymer inhibited sulfation of endogenous proteins. Mg2+ and Mn2+ promoted sulfation. 35S-Labeled proteins from monkey cerebellum and young (10 days old) rat brain were subjected to lectin-Sepharose chromatography to identify the presence of sulfated glyco-proteins. Labeled proteins from both these sources could bind and get eluted from Concanavalin A-Sepharose and Ricinus Communis agglutinin-Sepharose column suggesting the presence of mannose or galactose containing glycosulfoproteins.

Loss of Co2+ sensitivity of monkey brain cytosolic neutral alpha-D-mannosidase by cyclic AMP dependent phosphorylation.

alpha-D-Mannosidase that exhibits a pH optimum close to neutrality (neutral mannosidase) purified from monkey brain cytosol is known to be stimulated by Co2+ and this stimulation is suggested to be mediated through a Co2+ activated aminopeptidase that is inseparable from the neutral mannosidase and that cleaves amino acids from the neutral mannosidase. In the present studies, the phosphorylation on serine residues of the neutral mannosidase by cyclic AMP dependent protein kinase is demonstrated. After phosphorylation the mannosidase activity remained unchanged, but it was not stimulated by Co2+. The aminopeptidase activity, although it retained its response to stimulation by Co2+, showed a drastic reduction in its activity after phosphorylation. It is suggested that the loss of Co2+ sensitivity of the neutral mannosidase after phosphorylation is mediated through the aminopeptidase.

The characterization of a thermostable activator of beta-D-glucosidase in normal human saliva.

The existence of thermostable activators of beta-D-glucosidase (EC 3.2.1.21) in Gaucher tissues is known. We demonstrate the presence of a thermostable factor in normal human saliva which activates the beta-D-glucosidase of saliva. The activator is specific for beta-glucosidase. It is dialysable and susceptible to pronase digestion. Sephadex G-25 gel filtration indicates that the activator is of low molecular weight, and polyacrylamide gel electrophoresis shows the low molecular weight fraction to migrate as two closely moving protein bands. A major proportion of the activator does not bind to concanavalin A-Sepharose. The activator appears to act by binding to or modifying the salivary beta-glucosidase in a time-dependent manner.

Acid glycosidase and arylsulfatase activities of human cerebrospinal fluid as measured by concanavalin A-sepharose affinity chromatography.

An affinity chromatographic method using concanavalin A-Sepharose is described for the determination of N-acetyl-beta-D-glucosaminidase, arylsulfatase. alpha-L-Fucosidase and alpha-D-mannosidase activities in the human cerebrospinal fluid. By this method (starting with 12 to 20 ml samples of cerebrospinal fluid) the above enzymes could be obtained in a concentrated form and their activities could be determined within incubation periods of 30 min to 1 h under the assay conditions described. The pH optima of the enzymes were in the range of pH 4 to 5. About 80% of the total cerebrospinal fluid N-acetyl-beta-D-glucosaminidase was found to be the A form by DEAE-Sephadex A-50 chromatography. About 60% of the total arylsulfatase was also found to be the A form. Determination of these enzyme activities in a few samples of human cerebrospinal fluid indicated a rough proportionality between the enzyme activities and the protein concentration in the cerebrospinal fluid.

Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid.

Human cerebrospinal fluid acetylcholinesterase and butyrylcholinesterase. Evidence for identity between the serum and cerebrospinal fluid butyrylcholinesterase.

Human cerebrospinal fluid contained both acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) and they were estimated in the presence of selective inhibitors. Butyrylcholinesterase of human cerebrospinal fluid was similar to human serum butyrylcholinesterase in its electrophoretic mobility, glycoprotein nature and tyramine activation of the aryl acylamidase (EC 3.5.1.13) activity exhibited by butyrylcholinesterase. Moreover antibody raised against human serum purified butyrylcholinesterase could completely immunoprecipitate butyrylcholinesterase from human cerebrospinal fluid without affecting acetylcholinesterase. It is suggested that a useful method for the precise determination of acetylcholinesterase in human cerebrospinal fluid would be removal of butyrylcholinesterase by immunoprecipitation using antibody raised against human serum butyrylcholinesterase.

Aryl acylamidase activity in human erythrocyte, plasma and blood in pesticide (organophosphates and carbamates) poisoning.

Erythrocyte acetylcholinesterase (EC 3.1.1.7) and plasma pseudocholinesterase (EC 3.1.1.8) were determined from the day of admission up to 10 days in patients who have consumed organophosphate or carbamate poisons. In a number of patients, plasma pseudocholinesterase was completely inhibited on the day of admission but increased with the passage of days. Erythrocyte acetylcholinesterase was not completely inhibited and it also tended to increase with time in most cases. Patients in whom the erythrocyte acetylcholinesterase was very low and did not show an increase within the first few days expired indicating the prognostic importance of erythrocyte acetylcholinesterase. The profile of aryl acylamidase (EC 3.5.1.13) activity in plasma or erythrocytes showed a pattern similar to the respective cholinesterases. Moreover, whole blood aryl acylamidase activity was found to be a good index of erythrocyte acetylcholinesterase suggesting the prognostic usefulness of blood aryl acylamidase in the poisoned patients.

Phosphomannose binding proteins: the phosphomannosyl receptor and insulin like growth factor II receptor.

Proteins that bind phosphomannose residues in glycoproteins exhibit widely different functions. They are found as receptors of lysosomal enzymes, as ligatin that binds peripheral glycoproteins and as a lectin in parasites. The identity of the phosphomannosyl receptor for lysosomal enzymes and the insulin like growth factor II receptor raises interesting questions regarding their function.

Unusual catalytic activities and functions of cholinesterases.

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) have been shown to exhibit not only esterase activity but also an amine sensitive aryl acylamidase and a metallo-carboxypeptidase activities. There is also evidence to indicate that they have functions in the substantia nigra of brain, in neural cell differentiation, cell division and tumorigenesis, cell-adhesion and detoxication mechanisms. Butyrylcholinesterase is suggested to act as a back-up enzyme in acetylcholinesterase knock-out mice. Cholinesterases have catalytic or non-catalytic roles in these functions. Partial sequence homology to many other proteins having different functions and a metal binding site which can influence functions are probably factors that confer the non-cholinergic functions and activities on cholinesterases.

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion.

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.

Lysosomal enzyme binding receptor protein from monkey brain and its phosphorylation.

The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein.

Phosphorylation of casein, fibrinogen and calmodulin by a glycoprotein protein kinase from monkey cerebellum: a casein kinase II-like enzyme.

A glycoprotein protein kinase was isolated from monkey cerebellum by polylysine-Sepharose chromatography and affinity chromatography on Sepharose 4B coupled to the lectin, Concanavalin A. The protein kinase phosphorylated casein on serine and threonine residues and was stimulated by polylysine, polyarginine, spermine, histone, protamine and sphingosine, but was inhibited by heparin, poly (Glu, Ala, Tyr) and poly (Glu, Tyr). These characteristics were typical of casein kinase II. The protein kinase also phosphorylated fibrinogen and calmodulin and exhibited similar characteristics of stimulation by polylysine or polyarginine. The phosphorylation of fibrinogen (a glycoprotein), but not casein or calmodulin (non-glycoproteins), was significantly inhibited by Concanavalin A. Unlike casein kinase II, the enzyme did not undergo autophosphorylation. The collective results suggested that the enzyme from monkey cerebellum was a casein kinase II-like protein kinase and that phosphorylation of a glycoprotein substrate (fibrinogen) by the kinase could be influenced by a carbohydrate binding lectin.