Effect of phytohormones on pectate lyase activity in ripening Musa acuminata.

A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.

Pectate lyase activity during ripening of banana fruit.

Pectate lyase (PEL) activity was demonstrated in ripe banana fruits on supplementing the homogenizing medium with cysteine and Triton X-100. The enzyme was characterized on the basis of alkaline pH optimum, elimination of the activity by EDTA and activation by Ca(2+). PEL activity was not detected in preclimacteric banana fruits. PEL activity increased progressively from early climacteric and reached maximum level at climacteric peak and declined in post climacteric and over ripened fruits. Replacing pectate with pectin in PEL assay manifested enzyme activity even in preclimacteric fruits. In contrast to PEL, polygalacturonase activity progressively increased during fruit ripening even in postclimacteric fruits.

Enzymatic oxidant and antioxidants of human blood platelets in unstable angina and myocardial infarction.

In ischaemic heart conditions we report a remarkable increase in platelet xanthine oxidase activity and rise in the levels of malondialdehyde (MDA) with concomitant decrease in the activities of free radical scavenging enzymes - superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The increased levels of free radical generating system and MDA and lowered levels of free radical scavenging systems seem to have critical role in ischaemic heart conditions.

Purification and characterization of polygalacturonase from banana fruit.

Polygalacturonase isoenzyme 3 (PG-3) was purified to homogeneity with a specific activity of 0.7 mu katal mg-1 protein from banana fruit pulp. The purified enzyme was a glycoprotein with ca. 8% carbohydrate. The molecular weight of the native enzyme was found to be 90 +/- 10 kDa with a subunit molecular weight of 29 +/- 2 kDa. The enzyme exhibited optimum activity at pH 4.3 and temperature 40 degrees C with activation energy 35.4 kJ mol-1. A unique property of the enzyme was the requirement of -SH groups for the enzyme activity. The enzyme was inhibited by p-CMB and activated by 2-ME and DTT. The inhibition of p-CMB could be reversed by DTT. The enzyme contained eight free -SH groups. The Km of the enzyme was 0.15% for polygalacturonic acid.

Lipid peroxidation in human blood platelets during unstable angina and myocardial infarction.

A comparative study on the levels of lipid peroxidation in human platelets has been undertaken in patients of unstable angina and reperfused acute myocardial infarction and matched healthy individuals. Lipid peroxidation increases in these patients, most marked increase were in the patients of reperfused myocardial infarction.

Levels of glutathione reductase and glutathione peroxidase of human platelets in unstable angina and myocardial infarction.

Levels of glutathione peroxidase and glutathione reductase were measured in the platelets of 30 patients, 10 of them affected by unstable angina, 10 of them reperfused after myocardial infarction and 10 matched healthy controls. The specific activities of both the enzymes were lowered in both group of patients. Glutathione reductase activity resulted markedly lowered.

Multiple forms of polygalacturonase from banana fruits.

Three multiple forms of polygalacturonase (PG) in ripe and two in unripe banana (Musa acuminata) fruits were separated by DEAE-cellulose and further purified using Sephadex G-150 chromatography. The multiple forms can be differentiated from each other on the basis of their properties. PG1 and PG3 were identified as endo-PG and PG2 as exo-PG on the basis of decrease in viscosity, increase in reducing sugar and the reaction product. PG2 and PG3 increased with the ripening of fruits. PG1, PG2 and PG3 exhibited optimum activity at pH 3.3, 3.7 and 4.3, respectively. Complete loss of PG2 and PG1 activities occurred at 60 and 70 degrees, but PG3 retained 60 and 50% activity respectively. The three forms showed a different response towards divalent metal ions. Ca2+ activated PG1 activity only. Teepol 0.1%, inhibited PG1 activity by 25%, but PG2 and PG3 activities were completely inhibited. CTAB, 0.1%, had no effect on PG1 and PG2 activities, but inhibited PG3 activity by 40%. 2-ME stimulated PG2 and PG3 activities but had no effect on PG1 activity. Gel filtration through Sephacryl indicated M(r) of 23,200, 58,000 and 130,000, respectively, for PG1, PG2 and PG3. The substrate saturation curve for PG1 and PG2 were Michaelian, while PG3 showed biphasic curve. The Km values of PG1 and PG2 were 0.22% and 0.14%, respectively.

Physico-chemical and functional characterization of a high molecular weight carboxymethylcellulase from Cuscuta reflexa.

Carboxymethylcellulase (CMCase) was extracted and purified from an angiosperm parasite Cuscuta reflexa free from beta-glucosidase and other enzyme activities. The molecular mass and Stokes' radius of the purified enzyme are 144 kDa and 44 A, respectively. The diffusion coefficient and frictional ratio of the enzyme were 5.15 x 10(-7) cm2/sec and 1.27. The SDS-PAGE revealed homotetrameric nature of the enzyme with a subunit molecular mass of 35 +/- 1 kDa. Titration against DTNB and NBS revealed 19 sulfhydryl groups and 8 tryptophan groups, respectively, per mole of the enzyme. A sharp pH optimum at 5.0 was obtained. Cuscuta CMCase activity is unique amongst plant endoglucanases in being stimulated by Mg2+ and Mn2+ ions and by various thiols. Reaction product analysis, mode of enzyme action and substrate specificity test suggest the endo- nature of the purified CMCase. The enzyme showed K(m) value of 26 +/- 1 mg/ml for carboxymethylcellulose (sodium salt).

Purification and characterization of catalase from goat (Capra capra) lung.

Catalase plays a major role in the protection of tissues from toxic effects of H2O2 and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127 +/- 2 A. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with Ki values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.

Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone.

The N-deacetylation of thiacetazone, an antitubercular drug possessing hepatotoxic side effects, by an exclusively cytosolic arylacylamidase has been identified in the liver and kidney of rat by monitoring the appearance of its metabolite p-aminobenzaldehydethiosemicarbazone spectrophotometrically. Studies toward its characterization in liver cytosol revealed that the hydrolase possesses a broad pH optimum ranging from 6.0 to 9.0. The Km and Vmax values for the N-deacetylation of thiacetazone are 5.7 x 10(-4) M and 0.123 nmol of p-aminobenzaldehydethiosemicarbazone formed/min/mg cytosolic protein, respectively. The ability to metabolize thiacetazone was the same in the livers of cat, mouse and human, but lagged significantly in that of rat. Among the biodegradable esters examined as potential rivals of thiacetazone, only aspirin competitively inhibited thiacetazone hydrolysis (Ki = 2.1 x 10(-4) M). Discrimination of cytosolic thiacetazone N-deacetylase from nonspecific p-nitrophenylacetate esterase on the basis of their differential reactivity toward various inhibitors and activators disclosed that low concentrations of p-chloromercuribenzoate, AgNO3 and CuSO4 selectively undermine the activity of thiacetazone N-deacetylase, whereas SKF 525-A, ZnSO4 and FeCl3 are effective inhibitors of p-nitrophenylacetate esterase. However, divalent ions (Ca++ and Mg++) and EDTA failed to alter the activity of the enzyme. Besides, thiacetazone metabolism was significantly retarded upon exposure to malathion. Notably, Nal/Kl stimulated the N-deacetylase activity as a function of iodide concentration. The hydrolysis of thiacetazone in the liver and kidney remained uninduced by phenobarbital, 3-methylcholanthrene or benzo(a)pyrene (80 mg/kg, p.o., 8 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of chronic oral intake of cannabis extract on oxidative and hydrolytic metabolism of xenobiotics in rat.

Dietary intake of petroleum ether extract of cannabis leaves by rats in doses of 158, 250 and 500 mg/kg in the first, second and third week, respectively, caused selective induction of hepatic microsomal carboxylesterases/amidases without affecting the renal hydrolytic activity. Acetanilide N-deacetylase, p-nitrophenylacetate (NPA) esterase and acetylsalicylic acid (ASA) esterase I and II (active at pH 5.5 and 7.4) were stimulated 125, 64, 82 and 60%, respectively, whereas the activities of procaine esterase and acetylaminofluorene (AAF) N-deacetylase remained unaltered. The hydrolysis of acetylcholine was also unchanged. Upon withdrawal of treatment microsomal hydrolytic activity receded to basal levels within 7 days. Curiously though, the two-fold induction of thiacetazone N-deacetylase (118%), a cytosolic hydrolase, remained largely undiminished (62%). An appraisal of the hepatic cytochrome P450 mediated oxidative metabolism revealed approximately three-fold induction of aromatic hydrocarbon hydroxylase (AHH) metabolizing benzo(a)pyrene whereas the N-demethylation of aminopyrene was unaffected. These activities were restored to normal when resin administration was discontinued.

Purification and properties of goat liver catalase: two pH optima.

Goat liver catalase (EC 1.11.1.6) has been purified to homogeneity using the techniques of ammonium sulfate fractionation, DEAE-cellulose chromatography and gel-filtration through Ultrogel AcA-34 involving two alternating steps of column chromatography. The homogeneity of the purified enzyme was tested by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. The enzyme is a tetramer having a subunit molecular weight of 58,000 +/- 3000, contains six sulfhydryl groups per mole of the enzyme and shows pH optima at pH 6.8 and 7.7. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indoleacetic acid, cysteine, formaldehyde and sodium azide inhibit the enzyme non-competitively with Ki values of 4 +/- 1, 2.5 +/- 0.8, 6 +/- 1.5, 0.48 +/- 0.15 and 0.0013 +/- 0.0003 mM, respectively. Sulfhydryl group binding agents as well as thiol reagents inhibit the enzyme activity.

Fructose 1, 6 bisphosphatase activities in mouse liver as a function of age.

Neutral fructose 1, 6 bisphosphatase activity increases till 7 days, after which, a decline is observed postnatally upto 30 days. Alkaline fructose 1, 6 bisphosphatase follows the same pattern. The optimum activity of fructose 1, 6 bisphosphatase in mouse liver at pH 6.5 and 9.0 of all the periods, suggests the presence of both neutral and alkaline enzyme during the developmental period studied. On the basis of similarity observed in optimum pH, the same properties of enzyme at all the developmental stages studied, could not be ruled out.

Purification and physicochemical properties of starch phosphorylase from young banana leaves.

Starch phosphorylase from young banana leaves has been purified to homogeneity, as tested by disc polyacrylamide gel electrophoresis at various pHs and gel concentrations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophoresis, using the techniques of ammonium sulfate fractionation, DEAE-cellulose chromatography, and filtration through Sephadex G-100 and Sephadex G-200. The molecular weight of the enzyme is found to be 45000 as determined by gel filtration chromatography over Sephadex G-200. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having the molecular weight 55000. The enzyme contains eight SH groups per mol of the enzyme. Unlike other 1,4-alpha-glucan phosphorylases, no evidence is found for the presence of pyridoxal 5'-phosphate as a prosthetic group of the enzyme. Of the various amino acids tested, only aromatic amino acids inhibited the enzyme activity. ADP, AMP, and 3',5'-AMP did not produce any effect on the enzyme activity whereas ATP and UDP-glucose proved to be inhibitors. The enzyme utilized starch, amylose, and glycogen as primers with equal efficiency whereas dextrin, amylopectin, maltotriose, and maltose were less effective as primers. Schardinger dextrin, cellulose, or sucrose could not be utilized as a primer. The enzyme showed absolute specificity for glucose 1-phosphate as a substrate, and this could not be replaced by glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, or ribose 5-phosphate.