Effect of pH on Blepharisma intermedium. 5. Studies on esterases

Synopsis. Esterases of Blepharisma intermedium grown in buffered media of different pH values and in unbuffered media have been char­acterized. Selective inhibition studies indicate six carboxylesterases, two arylesterases and one cholinesterase in all cases. Although the type and number of the enzymes remain constant, there are variations in their inhibitor sensitivities

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol

Effect of pH on Blepharisma intermedium. 2. Cytochemical changes

No correlation was obsd. between fission and the concn. of chem. constituents and activities of some enzymes in B. intermedium. The cellular activities were governed by enzymes whose action was pH-​dependent. Changes in buffers, pH, and the molarity of the medium produced variations in the concn. of lipids, carbohydrates and proteins, in the activity of succinic dehydrogenase [9002-​02-​2]​, acid phosphatase [9001-​77-​8]​, and alk. phosphatase [9001-​78-​9]​, and in the shape, size, color, and motility of the organism. The internal pH of the organism affects metab., and is regulated by the extent of penetration of extracellular constituents across the cell membrane. The external pH, concn., and nature of the buffer constituents, including tris-​maleate-​Na hydroxide mixt., Na citrate, and Na acetate solns., det. the extent of penetration

Effect of pH on Blepharisma intermedium. 4. Changes in the activities of certain enzymes

Detn. of enzyme activities of B. intermedium grown in different buffered media of varying pH (5-​9) and molarity showed increases in acid phosphatase and glycogen phosphorylase, but decreases in alk. phosphatase, , esterase, and succinic dehydrogenase activities. These changes were obsd. under all condition of growth, compared to controls. The presence of alk. phosphatase in B. intermedium may be of significance, since the occurrence of this enzyme is irregular in ciliates

Purification and characterization of acetylcholinesterase isozymes from the latex of Synadenium grantii Hook, 'f'

Three acetylcholinesterase isoenzymes were purified from S. grantii latex by a combination of acetone fractionation, CM-​Sephadex C-​50 chromatog., Sephadex G-​200 gel filtration, and PE-​Cellulose chromatog. The homogeneity of the isoenzymes was established by PAGE and isoelectrofocusing. The pI values were 5.0, 5.2, and 5.4. The mol. wt. of each enzyme was estd. to be 70,​000 by a gel-​filtration method. SDS-​PAGE indicated that each enzyme consisted of 2 subunits of mol. wt. ∼35,​000. These enzymes were glycoproteins and were more sensitive towards carbamate inhibitors compared to organophosphates. They exhibited substrate inhibition and showed identical substrate specificity and inhibitor sensitivity. The rate consts. for different inhibitors were calcd. These 3 enzymes were considered to be charge isoenzymic forms of the latex acetylcholinesterase