Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT). Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts. The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase. Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis. The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur. During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway. The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme. Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA. However the ATP analogue cannot be incorporated into longer products than a trinucleotide. On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis. Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergendphospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate V , and the kinetic constant K:, were determined with the micellar substrate.The specificity of various substrates was examined by comparing the V,,,IK,B, values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.Keywords: phospholipase D ; phospholipid; mixed micelles; sunflower oil bodies.Phospholipase D (PLD) catalyses the hydrolysis of the ester linkage between phosphatidic acid and various alcohol moieties of several phospholipid species [I-31. This enzyme is widespread in the plant and animal kingdoms. It has been reported to play an important role in signal-transduction cascades in a variety of mammalian cells [4], where it appears to be activated by various hormones and neurotransmitters and some growth factors. In plants PLD is known to be involved in several cellular processes but its role has not been established clearly. The enzyme is present in the protein bodies of germinating mung bean cotyledons [5] and is associated with plasma and intracellular membranes in seedling tissues of castor bean [6]. Activity changes have been observed in conjunction with water stress [7], senescence [S] and pathogenic infection [9]. During mung bean seed germination and seedling growth, the decrease in the phospholipid content has been correlated with an increase in PLD activity [S]. In castor bean endosperm, immunoblot analyses have shown that the amount of PLD increases during the first five days of germination. It has therefore been suggested that these activity changes may be due to metabolic reactions involving membrane phospholipids, which are essential to plant growth and development [lo]. In rice bran, crude PLD prepara- (EC 3.1.4.4). tions were found to be able to react ...

The template properties of left‐handed synthetic polymers, the Z* form of poly[d(G‐C)] and the Z form of poly[d(G‐m5C)], have been investigated using an eucaryotic RNA polymerase, the class II enzyme from wheat germ. Results from a comparative kinetic study of transcription using the polynucleotide substrates in the B and Z conformations are reported. Optimal conditions for enzyme activity compatible with the preservation of the desired template conformation were determined. On the basis of several criteria, both physical (c.d. spectra of the polymers, sedimentability of the Z* form) and biochemical, it was demonstrated that the left‐handed conformations of poly[d(G‐C)] and poly[d(G‐m5C)] serve as templates for wheat germ RNA polymerase II. The level of incorporation was less than that exhibited by the B form of poly[d(G‐C)], the relative activity being a function of the precise experimental conditions. Activity ratios (Z*/B or Z/B) ranged from 0.1 to 0.5. The effect of various incubation parameters, including pH, salt concentration, temperature, and the presence of dinucleoside monophosphate primers were investigated. The Km values for nucleoside triphosphate substrates were slightly smaller for the Z* form of poly[d(G‐C)] than for the B conformation. Titration of DNA (Z* or B) with enzyme and reciprocal experiments suggested that the reduced activity of left‐handed templates might derive from the availability of fewer and/or lower affinity sites for initiation and/or translocation on these templates. Specific antibodies raised against left‐handed DNA strongly inhibited the observed transcription of Z* and Z DNAs by wheat germ RNA polymerase II.

A lipase from the latex ofEuphorbia chaaias was purified using a method involving extraction with apolar solvent and adsorption chromatography on silica gel. The lipase (specific activity, 1500 international units/mg ofprotein) was eluted from silica gel complexed with a lipid. The main protein fraction, which had a molecular mass of 38 kDa, was inactive when dissociated from the lipid fraction. When the lipid and protein fractions were reassociated, 72% of the lipolytic activity was recovered. This lipolytic activity was inhibited by diethylp-nitrophenyl phosphate, which was shown to bind the lipase with a molar ratio of 0.75. High specific activities (1000 international units/mg) were measured for the lipase of E. characias on lipid extracts rich in galactl diacylglycerols. The apolipase was sequenced up to residue 23. The B chain of ricin has a strong homology (43.5%) with that sequence and cross-reacted with antibodies raised against the purified lipase from E. characias. The activity of the B chain of ricin was comparable (54 international units/mg) to that of the apolipase of E. characias (100 international units/mg) mixed with the same lipid cofactor complex. The primary structure (residues 68-72) of the B chain of ricin contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly. Its reactivity with diethyl p-nitrophenyl phosphate indicates the presence of an activated serine that, in addition to its well-documented lectin activity for glactosides, suggests that the B chain of ricin may be a galactosyl diacylglycerol lipase, closely analogous to the lipase from E. characias. usually <0.1 pimol is hydrolyzed per min per mg). Much higher hydrolytic activities have been observed with the acyl hydrolases, which are not active on triacylglycerols, in various plant tissues (5). These activities are absent before germination and develop during the postgermination stage, concomitantly with the disappearance of the storage triacylglycerols (5). High levels of lipase activity, ranging from 7 to 30 jmol per min per mg (dry weight), were recently described in the fresh latex of branched nonarticulated laticifers belonging to the families Asclepia and Euphorbia (11). The lipolytic activity of all the latices was associated with sedimentable particles. Until recently attempts to solubilize the enzymatic activity from particulate fractions of latex have been unsuccessful, but we have succeeded in extracting and partially purifying a lipase from the latex of Euphorbia characias by using an apolar solvent extraction method (12). In the present paper, we report the purification to apparent homogeneity of the lipase from E. characias as an apoenzyme and describe some of its physicochemical properties, including its partial N-terminal amino acid sequence. Moreover, the B chain of ricin, which possesses (13) strong homology with this N-terminal amino acid sequence, was found to actively catalyze the hydrolysis of triacylglycerides. Data are presented here that document the association of a lipase activity with the B c...