Aggregation of banana pyrophosphate fructose 6-phosphate 1-phosphotransferase by glycerol.

Addition of glycerol during purification of banana (Musaceae, Musa cavendishii) pyrophosphate fructose 6-phosphate 1-phosphotransferase [(PFP), EC 2.7.1.90] initiated molecular aggregation of the enzyme. The aggregation process was dependent on the glycerol concentration. The native enzyme (66 kDa molecular mass) showed enhanced activity at 3% (V/V) or less of glycerol concentration. Glycerol concentration between 4 and 5% (V/V) affected a gradual and sequential aggregation of native form of the enzyme. These aggregated forms had molecular masses of 135, 200 and 270 kDa. The 135 and 200 kDa forms were stable for about 72 hrs and prolonged storage over 2 weeks resulted in the formation of the 270 kDa form. Concentration over 5% could reduce the time required for aggregation. Fru2.6 bis P activated the enzyme over ten fold, but did not help in the aggregation process. Studies on the role of glycerol on PFP specific activity suggested a difference in the activation process compared to that by Fru2.6bis P. Replacement of Hepes buffer by Tris increased the Fru2.6 bis P requirement for maximum activation by around 10 fold. Removal of glycerol from the buffer media resulted in almost complete inactivation of the enzyme.

The phenyl propanoid pathway enzymes in Solanum tuberosum exist as a multienzyme complex.

The elution profile of the core sequence enzymes of the phenyl propanoid pathway, namely phenyl alanine ammonia lyase, t-cinnamic acid 4-hydroxylase and p-coumaryl CoA ligase, on AcA 34 column suggested the existence of a high molecular form (P1) and a low molecular form (P2) for all the three enzymes. All the P1 forms eluted together in same fractions, while the P2 forms eluted out according to their respective molecular mass. Rechromatography of P1 form under identical conditions showed a similar elution profile (Q1 and Q2 forms). Further, the Q1 form did not show any significant increase in specific activity when compared to the P1 form. These results suggested the possibility of these enzymes existing as a protein cluster. Further confirmation was obtained on repeated column chromatography of the Q1 form in presence of 0.1 M KCl which did not result in complete dissociation of the complex to its individual enzyme components. The identification of the subunit polypeptide of the individual enzyme components in the multi enzyme complex and the in vitro demonstration of the phenyl propanoid core pathway reaction sequence using phenylalanine alone as a substrate supplementing the required cofactors for appropriate reactions substantiated that at least the core enzymes of the phenyl propanoid sequence existed as a multi enzyme complex.

Identification of a non-microsomal cinnamic acid 4-hydroxylase from potato tuber (S. tuberosum) and its partial purification.

The cytoplasmic localisation of cinnamic acid 4-hydroxylase (CA4H) has been shown by isolation and subcellular fractionation of the enzyme in Hepes buffer. The enzyme was purified by ammonium sulphate fractionation followed by AcA-34 molecular sieve chromatography. The enzyme existed as a high molecular mass which dissociated to a lower form on dilution on the column. The pH optimum, sulphydryl requirement, molecular and preliminary kinetic characteristics were investigated.

Studies on the induction of cinnamic acid 4-hydroxylase in potato tuber.

The change in activity of cinnamic acid 4-hydroxylase (CA4H) in potato parenchyma tissue exposed to various conditions has been examined. Maximum induction of CA4H activity was obtained at 18 hr of incubation. Though CA4H induction can occur in dark, over 100% increase in enzyme activity was obtained on exposure of the tissue to light. Actinomycin D and cycloheximide inhibited the induction process. Mn2+, though known to cause an induction of CA4H in Jerusalem Artichoke, strongly inhibited potato CA4H induction. Dithiothreitol enhanced the CA4H activity due to either activation or protection of the enzyme. CA4H induction was significantly regulated at very low concentrations of trans-cinnamate and paracoumarate.