Marcel Teissere

Improved purification and biochemical characterization of phospholipase D from cabbage

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.

Peroxidases associated with lentil root ribosomes

Abstract Three peroxidases are associated with the ribosomes of lentil roots. During cellular fractionation, the three haemoproteins are only present on these particles and not in the other fractions. The three peroxidases were isolated and obtained in a highly purified form. Their spectral characteristics and their molecular weights were determined. One is slightly acidic at pH 6·8, whereas the other two are slightly basic at this same pH. The incorporation of a 14 C-amino acid into these haemoproteins has enabled us to show that the basic peroxidases turn over rapidly whereas the acid peroxidase is very stable. Treatment of the roots with IAA considerably stimulates the de novo biosynthesis of the two basic peroxidases but not that of the acid peroxidase.

RNA polymerase III from wheat embryos Purification by affinity and hydrophobic chromatographies Characterization and molecular properties

It is now clearly established that higher plant cells contain diverse classes of DNA-dependent RNA polymerases of nuclear origin. Each class is thought to function similarly to its counterpart in other eukaryotes [ 11. Class B (or II) enzymes, which are generally the major components, have been purified from several plant sources and their subunit structure has been determined [2-51. Recently, the purification and the determination of the subunit structure of a class A (or I) RNA polymerase from soybean were achieved [6]. Class C (or III) RNA polymerase has not been characterized in most of the plant tissues so far investigated. However, we have previously reported that lentil roots [7] and wheat embryos [8,9] contain three types of DNA-dependent RNA polymerase activities: Ia and Ib which are insensitive to a-amanitin, II which is inhibited by low concentrations of this drug, and a third one which is 50% inhibited by 10 pg/ml of cy-aminitin and which could be tentatively identified with RNA polymerase III as defined for animals and lower eukaryotes [lo]. Nevertheless, to confirm the latter point it was necessary to compare the subunit structure of this enzyme with those of RNA polymerases I and II, for it has been reported that wheat germ would contain only enzymes I and II [ 111. In this paper, we describe a method of purifica-

Hormonal control of transcription in higher plants.

1. Nucleolar RNA polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated by a factor that changes the molecular properties of nucleolar RNA polymerase. 2. Four fractions, alpha, beta, gamma and delta, that stimulate the activity of RNA polymerase Ib, have been extracted from lentil roots. Two of them, gamma and delta have been studied. Factor delta can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well, while factor gamma is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors gamma or delta suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [gamma-32P]ATP and [3H]UMP in the RNAs synthesized in vitro. 4. Although the level of factor delta is independent of auxin treatment, that of factor gamma is doubled in auxin-treated roots. These results suggest that factor gamma is an auxin-induced protein that modulates the specific activity of the nucleolar RNA polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained by other workers.

Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds

Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.

Purification and properties of N-acetyl-β-d-glucosaminidase from Hevea brasiliensis latex

Abstract β-N-Acetylglucosaminidase from Hevea brasiliensis latex was isolated by anion exchange and gel filtration chromatography. The purified enzyme showed a single protein band on native electrophoresis. It is a glycoprotein with a molecular mass of 92 kDa, consisting of two 46-kDa subunits and has a glycosidic content of 16%. It shows optimal activity at pH 6.0 and at 50°C; its amino acid composition has been established. It is inhibited by several monovalent or divalent ions. The enzyme hydrolyses p- nitrophenyl -N- acetyl -β- d - glucosaminide (pNP-β- d -GlcNAc) with apparent Km and Vm values of 1.13 mM and 185 mM min−1 mg−1 of protein, respectively, at the optimum pH. p- Nitrophenyl -N- acetyl -β- d - galactosaminide (pNP-β- d -GalNAc) can also be used as a substrate but is less efficient. Glucosamine and glactosamine were competitive inhibitors with Ki values of 2.2 mM and 7.5 mM, respectively, at pH 6.0. The results of kinetic studies suggest that two ionizable groups with pK values of about 4 and 6.5 may take part in the reaction, possibly in the substrate binding. The vacuolar location of the enzyme is in agreement with the idea that the latex might play a lysosomal role in interacellular digestion processes.

Contribution a l'etude d'acides ribonucleiques a marquage rapide de la racine de lentille: Isolement et caracterisation, action d'une hormone sur leur synthese et sur celle de peroxydases

Abstract Chromatography on methylated albumin columns of RNAs from Lentil roots, shows the presence of rapidly labelled RNAs tenaciously bound onto the column and which cannot be eluted by high NaCl molarities. Elution at 35°, with sodium dodecyl sulfate, gives two peaks (α and β). At 70°, a third elution peak (γ) is obtained. For short labelling periods, RNAs of the α peak are uridine rich (UMP/AMP approx. 1.2), heterodisperse and may have an important percentage of high-molecular-weight species. RNAs from the β peak are adenine rich (UMP/AMP approx. 0.4). Their modal sedimentation constant is about 12 S. The mean life of the uridine-rich RNAs of the α peak is lower than 1 h; that of the RNAs from the β peak is higher than 2 h. RNAs from α peak are essentially, or exclusively, of nuclear origin. On the other hand, polysomic messenger RNAs are present in the β peak. A close similitude exists between uridine-rich RNAs described in animal cells and the nuclear rapidly labelled RNAs of Lentil roots contained in the α peak. Short treatment (45 min) of Lentil roots with indoleacetic acid increases the amount of nuclear uridine-rich RNAs. A prolonged treatment (5 h) causes an increase in various rapidly labelled RNAs, probably polysomic messenger RNAs. Treatment of excised roots with indoleacetic acid leads, after a lag period of about 5 h, to the synthesis of two peroxydases associated with the ribosomes. The mean life of their messenger RNAs is about 2.5 h.

Non-processive transcription of poly[d(A—T)] by wheat germ RNA polymerase II

RNA product distribution obtained during the transcription of poly[d(A—T)] by wheat germ RNA polymerase IIA under various experimental conditions was analyzed by high resolution polyacrylamide gel electrophoresis. Poly[r(A—U)] synthesis proceeded as if wheat germ RNA polymerase II was a non‐processive enzyme: a ladder of RNA products of increasing lengths was obtained, which apparently, terminated at every other nucleotide. RNA release was not dependent upon nucleoside triphosphate substrate concentrations. A likely explanation would be that ternary complexes enzyme: DNA: RNA were very much unstable; moreover, oligonucleotides released were not re‐used for further elongation by the enzyme.

Sequence similarity between heterogeneous nuclear RNA and polysomal messenger RNA in higher plants

The existence of heterogeneous nuclear RNA (HnRNA) in animal cells is now well documented [l-3] . Recent reports [4] present evidence for the occurrence of these molecular species in higher plants as well. It is firmly established [S] that the majority of these RNAs has a high turnover and never leaves the nucleus. Moreover, the possible precursor relationship of HnRNA to polysomal messenger RNA (mRNA) has been the focus of considerable interest [6,7] in recent years. The possible sequence similarity of these molecular species has been investigated mainly by the use of molecular DNA-RNA hybridization [7,8]. However, firm conclusions cannot be drawn from these studies because of the existence of reiterated sequences in the DNA of eukaryotic cells [9] . The aim of the present paper is thus to perform molecular hybridization experiments on plant DNA having no reiterated sequences in view of establishing a possible sequence similarity between HnRNA and polysomal mRNAs.

Transcription in vitro of Cauliflower Mosaic Virus DNA by RNA polymerase I, II and III purified from wheat embryos

Abstract RNA polymerases I, II and III purified from wheat embryos were used to transcribe CaMV DNA in vitro . With Mn++ as the divalent cation the efficiencies of transcription were respectively 50%, 20% and 90% for enzymes I, II and III. With Mg++ only enzymes II and III could use this template; enzyme I was unable to initiate RNA synthesis. Without ATP + GTP only enzyme III could form heparin-resistant initiation complexes; in their presence heparin-resistant RNA synthesis catalyzed by enzyme III is two-fold higher than that observed with enzyme II, suggesting that the formation of highly stable initiation complexes with double-stranded DNA is an intrinsic property of enzyme III.