Takekazu Horio

Possible partial reactions of the photophosphorylation process in chromatophores from Rhodospirillum rubrum

Abstract 1. 1. Chromatophores from Rhodospirillum rubrum were used to study the photosynthetic formation of ATP in relation to ATP-P 1 exchange, ADP-ATP exchange and ATPase. 2. 2. ATP-P 1 exchange was greatly stimulated by light (approx. 1000 ft-candles). Ascorbate affected the ATP-P 1 exchange activity in light in a similar manner to the photophosphorylating activity, both activities being greateest in the presence of 67 mM ascorbate. 3. 3. ADP-ATP exchange in light was stimulated more by PMS than by ascorbate. For both light ADP-ATP exchange and photophosphorylation, the optimal concentration of PMS was approx. 0.4 mM. 4. 4. In the presence of 3.3 mM ATP, ATPase activities in light and dark were depressed by the presence of more than 5 mM ascorbate. The inhibition by ascorbate was competitive with the neutralization caused by a high concentration of ATP. 5. 5. Photophosphorylation and ATP-P 1 and ADP-ATP exchange in light and dark were maximal at around pH 8, whereas ATPase activities in light and dark were maximal at pH 8.5 or more. 6. 6. Photophosphorylation activity and ATP-P i exchange and ATPase activity in light and dark were inactivated by prolonged sonication of the chromatophores. However, with prolonged sonication ADP-ATP exchanges with ascorbate in light and dark and with PMS in dark increased in activity, whereas ADP-ATP exchange with PMS in light was scarcely affected. 7. 7. ATPase activity was significantly stimulated by arsenate, 2,4-dinitrophenol and pyrophosphate, and depressed by quinacrine hydrochloride and quinine hydrochloride, even in the presence of arsenate and 2,4-dinitrophenol. 8. 8. ATP-P i and ADP-ATP exchange, which are induced by light in the presence of PMS (“in light” minus “in dark”: light-induced), were inhibited by quinacrine hydrochloride and 2,4-dinitrophenol to almost the same extent as photophosphorylation. 9. 9. ATP-P i exchange in dark was significantly inhibited by arsenate, quinacrine hydrochloride, quinine hydrochloride and 2,4-dinitrophenol, but ADP-ATP exchange in dark was not. The latter was markedly inhibited by pyrophosphate. 10. 10. Light-induced ATP-P i exchange with PMS was less inhibited by o -phenanthroline than photophosphorylation with PMS; in the presence of 0.67 mM o -phenanthroline, these two reactions were inhibited 10% and 48%, respectively. 11. 11. Photophosphorylation with ascorbate and light-induced ATP-P i exchange with ascorbate were inhibited by more than 1·10 −3 μg/ml of antimycin A. The minimal concentration required for complete inhibition was approx. 3.3·10 −2 μg/ml. In the presence of lower concentrations of antimycin A, light-induced ATP-P i exchange was inhibited less than photophosphorylation. 12. 12. From these findings, the roles of ATP-P i exchange, ADP-ATP exchange and ATPase activity are discussed in relation to the photosynthetic formation of ATP.

Resonance Raman study of the pH-dependent and detergent-induced structural alterations in the heme moiety of Rhodospirillum rubrum cytochrome c'.

The resonance Raman spectra and the structures of the heme moiety of Rhodospirillum rubrum cytochrome c were investigated for its five states characterized by absorption spectra; Types-a and -n of the reduced form and Types-I, -II, and -III of the oxidized form. The frequency of the ligand-sensitive Raman line suggested the coordination of lysine (Nepsilon) at the sixth position of the heme iron of Type-n. The sixth ligand of Type-III was deduced to be either lysine or histidine but would not be methionine. Type-a and Type-II gave the Raman spectra of rather normal high spin type but Type-I was unusual in the sense that the frequencies of the Raman lines associated primarily with methine-bridge CC-stretching vibrations were relatively high in comparison with those of other high spin hemoproteins. Type-I was converted directly to Type-III upon the addition of SDS or 2-propanol but the conversion occurred via Type-II when pH was increased. Structural difference between the high spin hemes of Type-I and Type-II was discussed in detail.

Growth inhibitors: Molecular diversity and roles in cell proliferation

Article faisant le point sur les connaissances actuelles concernant les inhibiteurs de croissance des cellules animales: mecanisme daction et proprietes fonctionnelles du TGF-β, du FGR (fibroblast growth regulator) et dautres dinhibiteurs de croissance

Site of photosynthetic electron-transport systems coupling phosphorylation with chromatophores from Rhodospirillum rubrum

Abstract 1. 1. With chromatophores from Rhodospirillum rubrum , phosphorylations coupled with photosynthetic electron-transport systems have been investigated. 2. 2. Under illumination with white light at a fixed high intensity, the addition of a strictly limited concentration of phenazine methosulfate was required to induce photophosphorylation at a maximal rate. The maximal rate of photophosphorylation was always much faster when induced by phenazine methosulfate than when induced by the other reagents tested such as ascorbate or succinate. The ratio of maximal rate of the phenazine methosulfate-induced to ascorbate-induced photophosphorylation varied from batch to batch of chromatophore preparations with experimental values from 2 to 5. 3. 3. The phenazine methosulfate-induced, and ascorbate-induced photophosphorylations were inactivated in a similar manner by illumination of chromatophores with ultraviolet irradiation and by additions of the following inhibitors: Na 2 SO 4 , quinacrine hydrochloride, 2,4-dinitrophenol, dicumarol EDTA and phenylmercuric acetate. Chromatophore suspensions, if resonicated or heated, lost the activity of ascorbate-induced photophosphorylation somewhat more rapidly than that of phenazine methosulfate-induced photophosphorylation. In the presence of an appropriate concentration of o -phenanthroline, the ascorbate-induced and succinate-induced photophosphorylations were inactivated completely, but the phenazine methosulfate-induced photophosphorylation was not affected. With the chromatophores treated with an appropriate concentration of Triton X-100, the ascorbate-induced and succinate-induced photophosphorylations were inactivated completely, whereas the phenazine methosulfate-induced photophosphorylation was inactivated only partially. 4. 4. The photooxidations of cytochrome c 2 , and cytochrome c were stimulated in rate by the addition of ADP. The stimulation by ADP was not significantly influenced in the presence or absence of P i . The photooxidation was stimulated by 2,4-dinitrophenol in the absence but not in the presence of ADP. The concentraton of 2,4-dinitrophenol required for the stimulation was nearly identical with that required for the inhibition of photophosphorylation. 5. 5. ATP was synthesized from ADP and P i , coupled with the photooxidations of cytochrome c 2 , and cytochrome c . The values of the P/O ratio obtained were less than one. 6. 6. Based on these findings, the site for phosphorylation coupled with the photosynthetic electron-transport systems are suggested.

Relationship between photosynthetic and oxidative phosphorylations in chromatophores from light-grown cells of Rhodospirillum rubrum

Abstract 1. Oxidation of NADH by molecular O 2 , phosphorylation coupled with the oxidation, and photophosphorylations induced by ascorbate, phenazine methosulfate and NADH, have been studied with chromatophores from light-grown cells of Rhodospirillum rubrum . 2. Chromatophores catalyze the oxidation of NADH aerobically in the dark. NADH oxidation is markedly stimulated by the addition of an energy-trapping system (ADP plus P i , or ATP-hexokinase-glucose plus P i ) over the whole range of pH; the rate is maximal at pH 7.4–7.8 in the absence of the energy-trapping system, and at pH 6.9 in its presence. 3. Coupled to NADH oxidation, ATP (or glucose 6-phosphate) is synthesized from ADP and P i . Mg 2+ , NADH, P i and ADP are each required to almost the same extent for both oxidative phosphorylation and photosynthetic phosphorylation with NADH; the rate is maximal in the presence of 5.0 mM Mg 2+ , and the values of K m are 33 μM for NADH, 1.3 mM for P i and 13 μM for ADP. The rate of oxidative phosphorylation with NADH is maximal at pH 6.8, while the rate of photosynthetic phosphorylation is maximal at pH 7.5 when induced by NADH, and at pH 8.0 when induced by ascorbate and by phenazine methosulfate. 4. o -Phenanthroline (0.1 mM) inhibits ascorbate-induced photophosphorylation by 80 % and NADH-induced photophosphorylation by 50 %, but does not significantly affect NADH oxidation and its coupled phosphorylation. These three reactions with NADH are completely inhibited in the presence of 0.01 M o -phenanthroline. Changes in the pH of the reaction mixture, or energy-trapping system, significantly influence the o -phenanthroline inhibition of photophosphorylation. Ascorbate-induced photophosphorylation, when assayed in the presence of hexokinase and glucose, is not appreciably inhibited by 0.1 mM. 5. When chromatophores are heated at appropriate temperatures, NADH oxidation and coupled phosphorylation are inactivated nearly in parallel with NADH-induced photophosphorylation, while ascorbate-induced and phenazine methosulfate-induced photophosphorylations are significantly more stable to heat. Preillumination with ultraviolet radiation inhibits NADH oxidative phosphorylation. 6. 0.01 M cyanide inhibits by 90 % both NADH oxidation and its coupled phosphorylation; the K m value for cyanide is approx. 0.7 mM. 7. Under anaerobic conditions, 1 μg/ml of antimycin A inhibits ascorbate-induced photophosphorylation completely and NADH-induced photophosphorylation up to 85 %. The activity of NADH-induced photophosphorylation still remaining in the presence of antimycin A is further inhibited by the addition of ascorbate; the K m value for ascorbate is approx. 0.2 mM, roughly equivalent to an E mid of +0.2 V. 8. Based on these findings, relationships between NADH oxidation, its coupled phosphorylation, and photophosphorylations are discussed.

Photosynthetic adenosine triphosphate formation and photo-reduction of diphosphopyridine nucleotide with chromatophores of Rhodospirillum rubrum

Abstract We have studied photosynthetic DPN reduction by succinate and ATP formation from ADP and P 1 in chromatophores of Rhodospirillum rubrum . Our findings are: 1. 1. Magnesium ion accelerates DPN photoreduction most effectively at 5·10 −3 M, but is inhibitory at higher concentrations. The stimulatory effect of the ion is similar to that on the photosynthetic ATP formation. 2. 2. Photoreduction of DPN is maximal in rate in the presence of 3·10 −4 M DPN, and is inhibited with increasing concentration. 3. 3. In response to variations in concentration of succinate or DPN, photoreduction of DPN reaches a steady state in which approximately half of the DPN present is reduced. 4. 4. DPNH/DPN (1:1) is oxidized by succinate/fumarate anaerobically in darkness, provided that the E h value of succinate/fumarate is more positive than −0.04 V. This accords with the suggestion that there is an enzyme system which catalyzes oxidation of DPNH by fumarate anaerobically in darkness. 5. 5. Photoreduction of DPN is inhibited in varying degree by various adenosine derivatives and pyrophosphate; i.e. strong inhibition with ATP, ADP and pyrophosphate; moderate with adenosine 3′-monophosphate, AMP and adenosine; none with orthophosphate. 6. 6. Photosynthetic ATP formation with “bound” ADP is inhibited by adenosine, adenosine 3′-monophosphate and DPN, but not by ADP or TPN. The presence of ATP or pyrophosphate inhibits ATP formation either with “bound” ADP or in the presence of exogenous ADP: The lower the concentration of ADP present, the greater is the inhibition. Kinetic data show that the inhibition by ATP is competitive with orthophosphate, but non-competitive with ADP, while pyrophosphate is competitive with both ATP and orthophosphate. Based on these findings, mechanisms for photosynthetic ATP formation and DPN reduction are suggested.

Novel protease bound with chromatins in normal and tumorous tissues of rats

Abstract A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma⪢ Rhodamine sarcoma≥ AH 130≥ thymus> spleen⪢ kidney≥ liver⪢ brain.

Purification and characterization of alkaline protease and neutral protease from chromatin of rats.

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.

Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect onin vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells

SummaryWe have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis.

Preliminary crystallographic study of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum

Crystals of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum were obtained with the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The crystal belongs to the cubic system, space group I432, with unit cell dimension a = 245.9 A. An asymmetric unit includes one-quarter (L2S2, L: large subunit, S: small subunit) of a hexadecameric molecule (L8S8, 544,000 Mr), which is located on the crystallographic point symmetry 222 or 4. The crystal diffracts to at least 3.0 A resolution.