G.G. Sanwal

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.

Goat liver catalase immobilized on various solid supports

Abstract Goat liver catalase has been immobilized on or in alumina, gelatin, polyacrylamide and hen egg shell. The egg shell-bound enzyme was more stable on storage, exhibited higher affinity for H 2 O 2 and was less sensitive to inhibition by formaldehyde and sodium azide compared to the soluble enzyme. Immobilized catalase can be reused several times without much loss of enzyme activity.

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.

Extraction of total phenolics in the presence of reducing agents

Abstract The incorporation of reducing agent during the extraction of plant tissues with ethanol and acetone yielded extracts with higher content of total phenolics reacting with Folin-Denis reagent.

Characterization of multiple forms of α-glucan phosphorylase from Musa paradisiaca fruits

Abstract Three forms of α-glucan phosphorylase from mature banana fruit pulp separated by ammonium sulfate fractionation and DEAE-cellulose chromatography were anodic at pH 8·6 on starch gel electrophoresis. The three forms differed in sensitivity to the phenolics extracted from immature and mature banana fruit pulp. Only two forms of the enzyme were detected in immature banana fruit pulp.

Enzymes of the banana plant: optimum conditions for extraction

Abstract The activities of starch phosphorylase, β-amylase, phosphohexoisomerase, acid and alkaline invertase, sucrose synthetase, sucrose phosphate synthetase and acid and alkaline phosphatase were determined in various parts of the banana plant, using homogenates prepared in media supplemented with polyvinylpyrrolidone or Triton X-100. The results indicated that the supplement of choice depended on the enzyme and the tissue under study.

Glucan phosphorylase in the leaves of Dendrophthoe falcata: Purification and characterization of enzyme

Abstract α-Glucan phosphorylase of the leaf tissues of D. falcata was partially purified and isolated in two forms, A and B, by DEAE-cellulose column chromatography. Both the forms utilized soluble starch with equal efficiency, the K m value being 0·12 and 0·25 g/l for A and B respectively. Form A phosphorylase utilized glycogen efficiently, with K m value of 0·42 g/l but glycogen did not serve as primer for the B form. In contrast, the B form alone could utilize achroic dextrin, though with less efficiency than starch. The K m values for glucose-1-phosphate were 5 mM and 1·7 mM for A and B. AMP activated phosphorylase A, at the optimum pH, but not the B form. Among other differences between the two enzyme fractions were stability towards heat, linearity of activity with protein concentration and response to added cations and mercaptoethanol. The two enzymes were sensitive to some phenolics; phloridzin, in particular, was highly inhibitory to fraction B, whereas fraction A was inhibited only slightly. The phenolics in the leaves of D. falcata were highly inhibitory to both forms of enzyme.

Intracellular localisation of hexokinase in Cuscuta reflexa

Abstract In Cuscuta reflexa 16% of the hexokinase activity was associated with the particulate fraction and the rest in the 105 000 g , 1 hr supernatant. In a sucrose gradient, hexokinase activity banded with an organelle at a mean density of 1.20 g cm −3 , coinciding with the mitochondrial marker, cytochrome c oxidase. Fractionation of isolated mitochondria by digitonin showed the presence of the enzyme in the outer membrane along with its marker rotenone-insensitive NADH cytochrome c reductase. No latent form of hexokinase was detected.

Multiple forms of α-glucan phosphorylase in banana fruits: Properties and kinetics

Abstract Multiple forms of α-glucan phosphorylase isolated from mature banana fruit pulp differ from each other in several respects—pH optimun, temperature optimum, energy of activation, primer specificity, kinetics, and sensitivity towards metal ions, nucleotides, sugar mucleotides, amino acids, glycolytic intermediates, sulfhydryl binding agents, sulfhydryl reagents and phenolics.

An allosteric α-glucan phosphorylase from banana fruits

α-Glucan phosphorylase (α-i, 4-glucan orthophosphate glucosyl-transferase EC 2.4.1.1) isolated from the pulp of mature banana fruit is an allosteric protein; its activity is inhibited by tyrosine (I0.5 = 1.6 mM) and ATP (I0.5 = 2.6 mM). The substrate saturation curve for Glc-i-P is changed from hyperbolic to sigmoidal in the presence of 3 mM tyrosine, which increases the S0.5 value for Glc-i-P from 4.5 to 6.0 mM. The substrate saturation curve in the presence of ATP is biphasic. The inhibitions by tyrosine and ATP were cooperative in nature. Freezing and thawing of the enzyme preparation led to desensitization with respect to inhibition by tyrosine