Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.

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.

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.

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.

Mammalian sulfoconjugate metabolism.

Sulfoconjugates occur ubiquitously as sulfopolysaccharides, sulfolipids and sulfoproteins. A variety of sulfotransferases catalyze the sulfation process with 3'- phosphoadenosine 5'-phosphosulfate as the sulfate donor. Sulfatases that catalyze the desulfation of different sulfoconjugates are known to be deficient in a number of genetic storage disorders.

The binding requirements of monkey brain lysosomal enzymes tο their immobilised receptor protein.

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind ß-D-glucosaminidase, α-D-mannosidase, α-Lfucosidase and ß-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4·5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an 'uncovered' phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature.

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P- 300 and by sodium dodecylsulphate gel electrophoresis was ~ 30,000. Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7·0 mol of inorganic phosphate per mol of protein. Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.