No abstract

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.Phosphoriboisomerase (EC 5.3.1.6) participates in the interconversion ofribose-5-phosphate and ribulose-5-phosphate in both the oxidative and reductive pentose phosphate pathways. We have now developed a rapid procedure for the isolation of the chloroplastic phosphoriboisomerase from pea (Pisum sativum) shoots. Although the enzyme has been purified from other sources (24) and spinach ( 18), this is the first time that the enzyme has been purified to apparent homogeneity from the chloroplasts of a higher plant.We have characterized the enzyme with respect to kinetic parameters and inhibitor constants, and we have determined the N-terminal sequence through 34 amino acids. Phase partitioning experiments indicate that the chloroplast phospho-'In memory of Professor Mordhay Avron.

Pea chloroplastic phosphoribulokinase nnd yeast phosphoribot~omera~e partition indel~ndently of one another in ta two.ph.~tse polyethylene$1~ol. dextran s~stem, btt[ apparent interaction is se,n when ribose.5.phosphate is added to the two.plntse system. It apl~ars that the p¢~ leaf kina~c recognizes ?east isoraerase when it is carryin$ metabolite,

Biosynthesis of triterpenoids (triterpene esters and triterpenols) by isolated latex ofEuphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid (0.55 nmol 100 p.1 latex 1 h-1 ) into triterpenoids was thirty times greater than acetate incorporation (0.02 nmol 100 41 latex 1 tn), indicating that the rate-limiting step in the pathway occurs prior to mevalonate. A particulate fraction, capable of converting mevalonate but not acetate into triterpenoids (15,000g pellet), showed a linear rate of triterpenoid biosynthesis over a period of four hours. No evidence was found to indicate that soluble latex proteins had an effect on either the biosynthesis or the removal of triterpenoids from this fraction. Electron micrographs of isolated E. lathyris latex showed the presence of latex particles and rod-shaped starch grains as well as a single-membrane-bounded structure which comigrated on Percoll gradients with the mevatonate to triterpenoids converting activity.Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme (0.02 nmol 100 p.1 latex -1 h.t) is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex.HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from 1 the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken fromCopaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessability to the different substrates and/or reflecting the metabolic controls on carbon aUocatibn to the terpenes.Mevalonate incorporation by Copaifera Iangsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. ACKNOWLEDGEM ENTSI thank Professor Melvin Calvin for providing me with the opportunity and the support to learn plant biochemistry in his labs. Members of the Calvin group not only helped me with my research but also ensured that it was not serious science at all times. I thank all those who spent time in the Calvin labs while I was there for their friendship, especially