Jinpei Yamashita

Observations on the nature of pulse-labeled RNA's from photosynthetically or heterotrophically grown Rhodospirillum rubrum

Abstract Messenger RNA (mRNA) was extracted from both photosynthetically and heterotrophically grown cells of Rhodospirillum rubrum pulse-labeled with [ 3 H]uracil and its properties were examined. Competition experiments in hybridization did not show any differences in base composition of the two RNAs. It is concluded that, if a specific light mRNA is required to mediate photopigment synthesis, it is present in undectectable amounts. The results suggest that, in the bacterial photosynthetic system, control of protein synthesis is effected at the translational, rather than transcriptional, level.

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

SummaryNADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg++, or Pi, Mg++, ATP and the hexokinase-glucose system. Kmvalues for Pi in NADH oxidation and phosphorylation are 10−3m and 8×10−4m, respectively. These Kmvalues are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg++, or Pi, Mg++, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 μg per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.

Inhibition of partial reactions in bacterial photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Abstract Inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) of the following partial reactions of bacterial photosynthesis has been examined using chromatophores prepared from light-grown Rhodospirillum rubrum: ascorbate- and PMS-induced photophosphorylation, NADH oxidation, NADH oxidatively coupled phosphorylation, NADH-cytochrome c2 reduction, succinate-NAD+ photoreduction, and anaerobic NADH oxidation by fumarate. All of these reactions were found to be inhibited by DCMU (and 3-(p-chlorophenyl)-1,1-dimethylurea) at concentrations in the 0.1 to 1.0 mM range. However, succinate-cytochrome c2 reduction, NADH-2,6-dichlorophenolindophenol reduction and soluble NADH: cytochrome c2 reductase were not inhibited. Based on these findings, it is proposed that DCMU and related compounds inhibit electron transport in chromatophores at a site(s) between NADH and either cytochrome b or a component on the reducing side of cytochrome b.

Purification and Properties of Polynucleotide Phosphorylase from Photosynthetic Bacterium, Rhodospirillum rubrum

1. Polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] was purified to near homogeneity from the photosynthetic bacterium, Rhodospirillum rubrum. The purified enzyme had a molecular weight of approximately 160,000, and consisted of two equivalent subunits of approximately 76,000 daltons. It catalyzed the three reactions described below. 2. In the exchange reaction of the beta-phosphate of nucleoside diphosphates with Pi by the purified enzyme in the presence of 3.3 mM Pi, 6.7 mMCl2, and 0.33 mM or 1.0 mM nucleotide at pH 8.0 and 20 degrees C, ADP, GDP, and CDP, and CDP were better substrates than UDP, while IDP and deoxyribonucleoside diphosphates hardly served as substrates. The ADP-Pi exchange activity was significantly inhibited by high concentrations of either ADP or Pi. 3. In the polymerization reaction of ribonucleoside diphosphates by the purified enzyme in the presence of 6.7 mM nucleotide and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, ADP was the best substrate; the activities relative to that with ADP were 55% with UD, 51% with CDP, and 48% with IDP, while GDP hardly served as a substrate, 4. In the phosphoryolysis reaction of polynucleoside diphosphates by the purified enzyme in the presence of 1.0 mM polynucleotide, 6.7 mM Pi, and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, poly[U] was the best substrate; the activities relative to that with poly[U] were 32% with poly[A], 28% with poly[I], 21% with poly[C], and 2% with yeast RNA, while poly[G] and yeast DNA hardly served as substrates. 5. The three kinds of activities of the purified enzyme described above were stimulated by divalent cations such as Mg2+, Mn2+, Cd2+, and Co2+.

Uracil incorporation and photopigment synthesis in Rhodospirillum rubrum

Abstract The relationships between bacteriochlorophyll and RNA synthesis in non-growing cells of Rhodospirillum rubrum were studied during transition from dark aerobic to light anaerobic metabolism. 1. 1. At high cell densities, uracil incorporation and bacteriochlorophyll synthesis were greatly stimulated by illumination. The light-stimulation of uracil incorporation occurred earlier. Cell mass and protein content did not increase significantly. 2. 2. The incorporated uracil was distributed in a slowly sedimenting (“slow”) RNA fraction during 1 h of incubation. The ribosomal and soluble RNAs were gradually labeled at later times. The rate of synthesis of the slow RNA was greater than those of the other RNA fractions particularly in the light. 3. 3. Sucrose-gradient centrifugation and methylated albumen kieselguhr column chromatography produced the same profiles for the pulse-labeled (3 min) and 60-min labeled RNAs. 4. 4. Mitomycin, chloramphenicol, antimycin A, 2,4-dinitrophenol, carbonylcyanide m- chlorophenylhydrazone , 2-n- nonylhydroxyquinoline -N- oxide and 5-fluorouracil inhibited both RNA and bacteriochlorophyll synthesis. Puromycin inhibited bacteriochlorophyll synthesis, but not RNA synthesis. 5. 5. It is concluded that under non-growing conditions during transition, the slow RNA fraction produced could contain a messenger RNA and that the possibility it includes a specific light messenger component requires further investigation of this fraction.

Affinities of Various Nucleases to DNA-Sepharose under Non-Digestive Conditions:Survey for Productive Affinity Chromatography

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pHs, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.