Search

It was previously found that rabbit serum contains a growth-inhibitory substance for a tumorigenic rat liver cell line RSV-BRL. In the present study, the growth inhibitor was purified from normal rabbit serum to show a homogeneous protein band with a molecular weight (Mr) of 56 k on SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The purified growth inhibitor, tentatively named rabbit serum-derived growth inhibitor (RSGI), potently inhibited the growth of RSV-BRL and nine kinds of other cell lines including three human tumor cell lines at a concentration of 20 ng/ml or higher. The growth-inhibitory effect of RSGI was reversible and appeared to be cytostatic rather than cytotoxic. RSGI was stable to heating at 56 degrees C for 30 min or treatment with 0.1 M 2-mercaptoethanol, but labile to heating at 100 degrees C for 3 min or treatment with 1 M acetic acid (pH 2.3), 6 M urea, 50% (v/v) 1-propanol, or 0.1% (w/v) trypsin. These properties of RSGI suggested that it was different from type beta transforming growth factors, tumor necrosis factor-alpha, and other known growth-regulatory factors.

1. DNase I from porcine pancreas, if Mg2+ was present, hydrolyzed both sDNA and dDNA, whether free or bound to Sepharose. The hydrolysis rates were maximum at pH 7.5 with the bound DNAs and at pH 7.0 with the free DNAs negligible at pH 4.0 and pH 10.5 with the free and bound DNAs. The hydrolysis was completely inhibited by 50 mM sodium citrate. 2. With 50 mM citrate buffer (Ph 4.0), DNase I was effectively adsorbed on the DNA-Sepharoses in the absence of 5 mM Mg2+. The adsorbed enzyme was effectively eluated by the buffer containing 1 M KCl (eluate). The amounts of the eluated enzyme were approximately 1.5 X 10(5) units/mg DNA with sDNA-Sepharose and approximately 3.0 X 10(5) units/mg DNA with dDNA-Sepharose. This simple adsorption-elution of the pancreas extract resulted in approximately 300-fold purification of DNase I with a yield of 95%. In the elute, the ratios in activity of trypsin, chymotrypsin and RNase to DNase I were 1/(4.0 X 10(5)), 1/(5.3 X 10(3)), and 1/(4.1 X 10(2)) as low as in the extract, respectively. In addition, the eluate was not contaminated by kallikrein or carboxypeptidases A and B. 3. Upon repeating the adsorption-elution described above, the adsorbing capacities of DNA-Sepharoses gradually deteriorated with the whole pancreas extract, but not with the precipitate of the extract formed on 60% ammonium sulfate saturation, which contained 90% of the DNase I. With the precipitate, one dDNA-Sepharose solumn was repeatedly usable at least 20-times without deterioration. The DNase I preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis. 4. Conceivably, the above-mentioned adsorption of DNase I on DNA-Sepharoses was mainly due to the steric and electrostatic affinity of a relatively large moiety of the DNA molecule to the substrate-binding site, but not to the catalytic site, of the enzyme.

It was previously reported that in chromatophores of Rhodospirillum rubrum, reaction center, which consists of three kinds of protein (Mm, about 78K), is a small fragment of a large protein complex (PRU; photoreaction unit), which contains six other kinds of protein including light-harvesting bacteriochlorophyll protein, has Mm of about 700K and is free of phospholipid [J. Biochem. 86, 1211-1224 (1979); 94, 1815-1826 (1983(]. In the present study, the photosynthetic, cyclic electron transport system sensitive to antimycin A was effectively reconstructed by incubating 60 nM PRU (which contained 1 mol of reaction center and 2 mol of ubiquinone-10 per mol) with 300 nM each of oxidized ubiquinone-10 protein, reduced cytochrome c2 and lipoamino acid (which were all purified from Rhodospirillum rubrum) in the presence of low concentrations of cholate and deoxycholate (pH 8.0). In the light, the cytochrome was oxidized while the quinone was reduced. The oxidation and reduction each progressed rapidly at first, then slowly, reaching maxima (steady states) 1-2 min after the light had been turned on. At the steady states, 30% of the cytochrome was oxidized while 11% of the total quinone was reduced. When the light was turned off, the original oxidation-reduction states of the cytochrome and quinone were restored at rapid rates initially then at slow rates. Antimycin A stimulated the slow rates in the light-on state and depressed them in the light-off state, but did not influence the fast rates. Ubiquinone-10 protein was required for the antibiotic-sensitive, slow oxidation reactions. This indicates that the slow rates were due to cyclic electron transport. Cytochrome c2 was tightly bound to PRU at a molar ratio of 1:1. This cytochrome as well as the quinone bound to PRU was responsible for the fast rates. PRU had other sites able to bind cytochrome c2 and ubiquinone-10 protein with Km of 0.4 and 0.1 microM, respectively. Of the polar lipids tested, lipoamino acid was the most effective for reconstruction, and its effect was maximal at 300 nM, which is far below its critical micelle concentration.

The amino acid sequence of an [8Fe-8S] ferredoxin isolated from the culture medium of Rhodospirillum rubrum, a photosynthetic purple non-sulfur bacterium, was determined by a combination of various conventional procedures. The sequence was A-Y-K-I-E-E-T-C-I-S-C-G-A-C-A-A-E-C-P-V-N-A-I-E-Q-G-D-T-I-F-V-V-N-A-D-T-C-I-D-C - G-N-C-A-N-V-C-P-V-G-A-P-V-A-E (55 amino acid residues). It lacked methionine, leucine, histidine, arginine, and tryptophan. The molecular weight was calculated to be 5,568 excluding iron and sulfur atoms. The distribution of 8 cysteine residues was exactly the same as that of clostridial-type ferredoxin, suggesting retention of the duplication of the bacterial ancestral ferredoxin gene. The extracellular ferredoxin of R. rubrum was compared with other ferredoxins observed in closely related photosynthetic bacteria and the evolutionary significance of this ferredoxin is discussed.

Glycerol dehydrogenase [EC 1.1.1.6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20 mM acute buffer (pH 6.0) on either octyl-Sepharose or 10-carboxydecyl-Sepharose, among ten different kinds of n-alkyl-Sepharose derivatives tested, some of which have an amino or a carboxyl group as a terminal residue. The enzyme adsorbed on 10-carboxydecyl-Sepharose is 95% eluted with 0.26 M NaCl. n-Propanol (10% and 15%, but not 5%), and various nucleotides such as NAD+, NADH, NADP+, NADPH, AMP, ADP, and ATP (1 mM) are also effective for elution. The elution with nucleotides is facilitated by 5% n-propanol. On the other hand, the enzyme adsorbed on octyl-Sepharose is not eluted under the conditions described above. These results suggest that the adsorption-elution of the enzyme on 10-carboxydecyl-Sepharose is due to a combination of hydrophobic interaction and electrostatic repulsion between a specific locus of the enzyme surface and the 10-carboxydecyl residue. Experimental conditions are described under which the enzyme can be purified 266-fold with a yield of 79% by a single chromatography of the cell extract on a 10-carboxydecyl-Sepharose column.

Polarographic studies on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum were carried out at 24 degrees. 1. Using a carbon-paste electrode as the working electrode, polarographic waves characteristic of oxidation-reduction components were observed in the presence, but not in the absence of Triton X-100; these waves were therefore measured in the presence of the detergent. 2. At least two kinds of oxidation-reduction components were detectable, having different half-wave potentials (E1/2); at pH 7, one had an E1/2 value of +275 mV (POC+275) and the other had a value of +60 mV (POC+60). 3. POC+275 was reduced by succinate and by NADH. Both reductions were almost completely inhibited by antimycin A, which hardly affected the reductions of ubiquinone-10 by succinate and by NADH. Most POC+275 molecules were not reduced by the substrates when quinones were extracted from the chromatophores, and the reductions were mostly restored when ubiquinone-10 was re-added. This indicates that POC+275 is functional between ubiquinone-10 and cytochrome c2 in the electron transport system. 4. POC+60 was reduced by succinate, but hardly at all by NADH. The reduction of POC+60 was not influenced either by the addition of antimycin A or by the extraction of quinones. This suggests that POC+60 is functional in the process from succinate dehydrogenase [EC 1.3.99.1] to ubiquinone-10 in the electron transport system. 5. Of the POC+275 reducible by dithionite, approximately 70% could be reduced in the absence of Triton X-100, provided that the potential of the working electrode immersed in chromatophore suspensions was set at potentials of 0 mV or lower and that the electrochemical reaction was carried out at pH 7.5. When the potential of the electrode was set at +50 mV (the same as the E1/2 value of ubiquinone-10 bound with chromatophores), and the suspension was allowed to stand for various lengths in the presence of the detergent, it was found that approximately half of the electrochemically reducible POC+275 was rapidly reduced, followed by a slow reduction. The discrepancy in the oxidation-reduction equilibrium on the basis of the E1/2 values of ubiquinone-10 and POC+275 is discussed.

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+.

Reaction center of chromatophores of Rhodospirillum rubrum consists of three kinds of protein, H-, M-, and L-subunit, and is bound with many other kinds of protein to form a larger protein complex (PRU; photoreaction unit), which contains all the bacteriochlorophyll. In the present study, purified PRU was dissociated in a stepwise manner in the presence of various mixtures of lithium dodecyl sulfate, sodium cholate and/or sodium deoxycholate, and separated into five, smaller protein complexes (PL1, PL2, PL3, PL4, and PL4') by high-speed molecular-sieve chromatography. The protein complexes were analyzed for molecular mass (Mm), protein composition, and molecular weights of the constituent proteins by the chromatography described above and by lithium or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggest that PRU consisted of 1 molecule each of 40K, 39K, 31K (H-subunit), 25K (M-subunit), and 22K (L-subunit), about 12 molecules each of 12K (light-harvesting bacteriochlorophyll-protein) and 11K, and about 6 molecules each of 10K and 9K (the protein nomenclature refers to the apparent molecular weights); the measured and calculated Mm values were 650K and 547K, respectively. The compositions of the other protein complexes were as follows. PL1 = PRU-10K-9K (measured & calculated Mm, 520K & 409K); PL2 = PL1-39K (340K & 267K); PL3 = PL2-40K (160K & 147K); PL4 = PL3-31K-25K (90K & 82K); PL4' = 31K + 25K + 22K (inactivated reaction center) (90K & 78K). The molar ratios of 12K and 11K to 25K were lower in the dissociated protein complexes than in PRU, and they differed from one complex to another. The locations of the constituent proteins in PRU are discussed.

With Rhodospirillum rubrum, hydrogenase was found to exist partly as an extracellular enzyme in the culture medium. After 4-day cultivation, the total activity and the specific activity of the enzyme in the medium were about 10 times and 230 times as high as those in the crude extract obtained from disrupted cells. The time course for the production of hydrogenase during cultivation was studied.

Polyclonal antibodies were prepared to the subunits of the spinach photosystem II fraction (PS II): p47, p43, p27, p33, p24, and p17. (The protein nomenclature refers to Mr). p47 and p43 are the subunits of reaction center complex, and p27 is light-harvesting chlorophyll protein. p33, p24, and p17 are extractable from PS II with 1 M Tris, and p24 and p17 with 1 M NaCl. With untreated PS II fractions, the antibody to p24 inhibited the photosynthetic oxygen-evolving activity, but not the DCPI-photoreduction activity in the presence of DPC, indicating that p24 played an important role in the former activity. Bindings of the respective antibodies to the PS II treated with sodium dodecyl sulfate were regarded as 100%. To untreated PS II, the bindings were 20-30% for p47, p43, and p27, about 50% for p33, and 70-80% for p24 and p17. To NaCl-washed PS II, the binding to p33 increased by 9%, indicating that p33 was adjacent or bound with p24 or/and p17. To Tris-washed PS II, the binding to p43 increased by 7%, indicating that p43 was adjacent or bound with p33. To PS II treated with 3% of Brij 58, only the binding to p27 increased appreciably. To PS II treated with 1% of octyl glucoside, the binding to p47 was still lower than 50%, whereas those to the other subunits were 74-91%. These values could be a measure of the extents to which the subunits were exposed to the aqueous phase, because of the nature of polyclonal antibodies. These results suggest that in intact PS II, p47, p43, and p27 were in most part buried in the inside, p47 being located at the most central and p27 at the outermost part, whereas p33, p24, and p17 were exposed to the outside by 50-75%.

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 pH's, 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.

It is known that the coupling factor purified from the acetone powder of chromatophores from Rhodospirillum rubrum shows ATPase activity in the presence of Ca(2)+, but not in the presence of Mg(2)+ or Mn(2)+. The present study deals with conditions, under which the Ca(2)+-ATPase activity is reversibly converted into Mg(2)+- and Mn(2)+-ATPase activites with the purified coupling factor. 1. Of the pH indicators tested, 6 kinds coverted the Ca(2)+-ATPase activity into Mg(2)+- and Mn(2)+-ATPase activities in the order, ethyl orange greater than tropaeolin 000 greater than or equal to metanil yellow greater than tropaeolin 00 greater than ethyl red greater than or equal to bromthymol blue. 2. Of the detergents tested, those other than Triton X-100 and Brij 58 caused the conversion described above; dodecylsulfonate was most effective, whereas dodecylpyridinium chloride was moderately effective. 3. 2,4-Dinitrophenol stimulated approximately two-fold the Ca(2)+-ATPase activity, but not the Mg(2)+- or Mn(2)+-ATPase activity at all. However, in the presence of dodecylpyridinium chloride, the pH indicator remarkably stimulated the Mg(2)+- and Mn(2)+-ATPase activities, accompanied with a partial inhibition of the Ca(2)+-ATPase activity. Methyl red and ethyl red showed similar effects. 4. All the nucleoside triphosphates tested can serve as the substrate. ATP was most effective for the Ca(2)+-ATPase activity, whereas dATP was most effective for the Mg(2)+- and Mn(2)+-ATPase activities induced by ethyl orange. 5. In the presence of ethyl orange, the ATPase activity was induced by various divalent cations in the following order of effectiveness, Mg(2)+ greater than Zn(2)+ greater than CO(2)+ greater than Mn(2)+ greater than Ni(2)+. 6. The mechanism of the reversible conversion from the Ca(2)+-ATPase activity to the Mg(2)+- and Mn(2)+-ATPase activities by pH indicators and detergents is discussed.

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.

When the photoheterotroph, Rhodospirillum rubrum, was grown in the light, ferredoxin was excreted from the cells in a significant amount, as well as hydrogenase. The extracellular ferredoxin was purified to a homogeneous state. The molecular weight was approximately 9,000, and the oxidation-reduction mid-potential was -0.29 V (N=1) at pH 7.0 and 25 degrees C. The amino acid composition was different from those of the intracellular ferredoxins, which were already known. The contents of non-heme iron and acid-labile sulfur were 10.6 and 7.9 mol/mol protein, respectively. The extracellular hydrogenase catalyzed the evolution of hydrogen gas from the ferredoxin in the reduced form. The Km for the ferredoxin was 4.1 micro M, one-seven hundredth as low as that for methyl viologen. There is a possibility that hydrogenase here were functional for evolution of hydrogen gas outside the cells.

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.

1. The light-induced pH change of chromatophore suspensions from Rhodospirillum rubrum was stimulated significantly and similarly by KCl, NaCl, LiCl, RbCl, CsCl, MgCl2, MnCl2, and CaCl2. In the dark, the pH of chromatophore suspensions decreased immediately and markedly on adding these salts. 2. The light-induced pH change stimulated by KCl plus valinomycin was inhibited by LiCl and NaCl, but not by RbCl. 3. The optimum pH values for light-induced pH change and photosynthetic ATP formation were around 5 and 8, respectively. The amount of chromatophore-bound ubiquinone-10 reduced in the light was independent of pH from 5 to 9. At pH 8, the number of protons incorporated into chromatophores in the light was one-half of the number of ubiquinone-10 molecules reduced in the light. 4. Among several pH indicators tested, bromothymol blue (BTB) and neutral red (NR) showed absorbance changes on illumination of chromatophores. Although the pH change indicated by the absorbance change was opposite to the light-induced pH change of the medium, the effect of KCl on the absorbance changes of BTB and NR, and the effect of valinomycin on that of NR, but not on that of BTB, were similar to those on the light-induced pH change. 5. The light-induced absorbance change of BTB was significantly inhibited by NR, whereas that of NR was hardly influenced by BTB. 6. Oligomycin stimulated the light-induced absorbance change of BTB under either non-phosphorylating or phosphorylating conditions. On the other hand, that of NR under phosphorylating conditions was 50% of that under non-phosphorylating conditions, and was increased by oligomycin.

We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.

1. The chlorophyllase [EC 3.1.1.14] purified from greened rye seedlings hydrolyzed the bacteriochlorophyll isolated from Rhodospirillum rubrum, but not the pigment bound to the membrane of chromatophores or spheroplasts from the bacterium. 2. Acetone, if added at such concentrations that the bound bacteriochlorophyll would not be solubilized, enabled the enzyme to hydrolyze the bound pigment. The acetone concentrations required for half the maximum hydrolysis rates were 16% with chromatophores and 7% with spheroplasts. 3. The enzymic hydrolysis of the bound bacteriochlorophyll in the presence of acetone removed bacteriochlorophyllide from the membrane, leaving its esterifying alcohol, possibly all-trans-geranylgeraniol, in situ. 4. Washing of chromatophores with 30% acetone removed about 10% of the bound bacteriochlorophyll. The bound pigment remaining after washing was not hydrolyzed by the enzyme unless acetone was added. 5. It seems possible that light-harvesting bacteriochlorophyll was mostly, if not all, bound to the inner surface of chromatophores (the outer surface of spheroplasts), having its esterifying alcohol residue buried in the membrane and its porphyrin residue emerging from the membrane into the inside solution; thus, chlorophyllase could not make contact with the ester linkage between the esterifying alcohol and porphyrin moieties of the pigment unless the esterifying alcohol residue was partly exposed.

1. All the water-soluble yeast enzymes tested, which were only partially precipitated at best in the presence of high concentration of salts such as ammonium sulfate or sodium formate, were adsorbed on a column of cellulose in the presence of the same concentrations of the salts, and the adsorbed enzymes were chromatographically eluted by decreasing the concentration of the salts. 2. Even in the presence of high concentration of the salts, the adsorbed enzymes were eluted by urea or by "hydroxy-rich" reagents such as sucrose. 3. Under the experimental conditions used, the salt concentrations required for elution of the adsorbed enzymes were lower with cellulose than with DEAE-cellulose, CM-cellulose, or P-cellulose, indicating that ion exchange groups, either cationic or anionic, affected the adsorption, although the ion exchange groups of DEAE-cellulose, CM-cellulose, and P-cellulose were weakly but definitely functional as ion exchangers even in the presence of high concentrations of the salts. 4. The principal attractive force between cellulose and the enzyme was deduced to be due to hydrogen bonding. 5. This hydrogen bond chromatography was applied for the purification of some yeast enzymes.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).

Peroxidase was prepared from extracts of barley leaves and separated into seven components, different in pI. The purification procedure comprised two parts. The first part was based on the fact that all the components had practically the same molecular weights. It consisted of fractionations with acetone and ammonium sulfate, ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose CL-6B, and molecular-sieve chromatography on Ultrogel AcA44; the components were all purified together to near homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the procedure resulted in 1,200-fold purification with a yield of 39%. The ion-exchange chromatographies were carried out under conditions such that the components would not be adsorbed. In the second part, the enzyme preparation was separated into the seven components by repeating isoelectric electrophoresis. Their isoelectric points (pI) were 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. The components other than the pI 6.3 and 6.8 components were each purified to homogeneity in the electrophoresis. The seven components thus prepared were the same in molecular weight on SDS-gel electrophoresis (44,000) and showed absorption maxima at the same wave-lengths (403, 496, and 534 nm), RZ (A403/A275) ranging from 2.09 to 2.81. Their protoheme IX contents were 0.81-1.07 mol/mol, and their true sugar contents 15-26% (g/g). The amino acid compositions suggest that the five components described above are not real isoenzymes, but exhibit different pI values due to differences in glycosyl residue. The pI 9.3 component was crystallized in spite of its high sugar content.

It was found that a non-tumorigenic epithelial cell line from the liver of a Buffalo-strain rat (BRL) secreted into the culture medium various inhibitors of the growth of BRL and RSV-BRL (tumorigenic BRL transformed by infection of Rous sarcoma virus). The secreted inhibitors were classified into two types: one inhibited the growth of BRL to a greater extent than that of RSV-BRL (non-tumorigenic BRL growth inhibitor, NGI), and the other, vice versa (tumorigenic BRL growth inhibitor, TGI). Two NGI (NGI-I and NGI-II) and two TGI (TGI-I and TGI-II) were highly purified from the serum-free conditioned medium. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis without 2-mercaptoethanol, NGI-I and II gave protein bands with molecular weights (Mr) of 56,000 and 21,000, respectively. TGI-I and II gave a band that migrated faster than bromophenol blue marker dye, but they did not pass through an ultrafiltration membrane with an Mr cutoff of 5,000. In the presence of a reducing reagent, only NGI-II showed a decrease of Mr, from 21,000 to 11,000. NGI and TGI showed 50% growth inhibition with BRL and RSV-BRL, respectively, at 5-15 ng/ml in the medium containing 10% fetal calf serum. NGI and TGI all were stable to 1 M acetic acid (pH 2.3) and 6 M urea, but labile to 5 mM dithiothreitol or trypsin. Of the eight cell lines tested, NGI-I was most effective on BRL, NGI-II on BRL and HSC-3 (human tongue squamous carcinoma), and both TGI-I and II on RSV-BRL.

It is known that the halophilic green alga Dunaliella tertiolecta grows under hypertonic conditions (with NaCl), which induce the intracellular accumulation of high concentrations of glycerol in order to counterbalance the osmotic change. The effects of NaCl and glycerol on the photosynthetic oxygen-evolving activity of thylakoid membranes prepared from D. tertiolecta were investigated in relation to the dissociation of the membranes. It was found that proteins with Mr of 24,000, 17,000, and 13,000 were dissociated from thylakoid membranes of D. tertiolecta by washing with 1 M NaCl, whereas the photosynthetic oxygen-evolving activity was stimulated 2-fold by 0.1-1.5 M NaCl. The antibodies against spinach 24K and 17K proteins did not cross-react with Dunaliella 24K and 17K proteins, respectively. The salt-tolerant feature of the oxygen-evolving activity with Dunaliella thylakoid membranes may be related to the difference of the properties of these two proteins between D. tertiolecta and spinach. When the membranes were washed with 1 M Tris, proteins with Mr of 50,000 and 31,000 were also dissociated in addition to the 24K and 17K proteins described above. The antibody against spinach 33K protein cross-reacted with 31K protein of D. tertiolecta, showing that Dunaliella 31K protein corresponds to spinach 33K protein. When the membranes were treated with a mixture of 1% cholate and 2% deoxycholate, the oxygen-evolving activity was completely depressed, but the depressed activity was significantly restored by organic solvents. Glycerol and dimethylsulfoxide were the most effective for the restoration.(ABSTRACT TRUNCATED AT 250 WORDS)

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.

1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.

Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode.

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.

It was found that the cytoplasm of light-grown cells of Rhodospirillum rubrum could catalyze the reduction of methyl viologen (MV) (Em, 7 = -0.44 V) by NADH and NADPH. In the present study, the enzyme capable of catalyzing MV reduction by NADH (NADH-MV reductase) was purified 1,500-fold from an extract of cells with a yield of 4.4%. The purification procedure comprised (NH4)2SO4 fractionation, and chromatographies on Sepharose CL-6B, DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Blue-Cellulofine, and TSK-Gel G3000SW. Two NADPH-MV reductases were separated during the purification. The NADH-MV reductase obtained was nearly homogeneous, as judged on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The enzyme has a molecular weight of 220,000 and an isoelectric point of 4.8; it is composed of four subunits with a molecular weight of 57,000, and is bound with about 1 mol FAD/mol subunit. The activity is optimum at pH 8. The Km values for NADH and MV are 115 microM and 1.3 mM, respectively, with a molecular activity of 13,000 min-1. The activity was stimulated 2.4-fold in the presence of 20-100 mM ammonium ions. The enzyme also catalyzed the reduction of benzyl viologen, methylene blue and 2,6-dichlorophenol-indophenol (Em, 7 = -0.36, +0.011, and +0.217 V, respectively) at comparable rates. The ratios of the activity with NADH to that with NADPH were 80, 133, 41, and 5.5 with MV, benzyl viologen, methylene blue and 2,6-dichlorophenolindophenol, respectively. The enzyme was significantly stable in the presence of both 5mM 2-mercaptoethanol and 20% (w/v) glycerol. The activity was not appreciably influenced by the presence of 2 M urea, although the reagent caused dissociation to the subunits.

Hydrogenase was easily solubilized from light-grown cells of R. rubrum with 10 mM Na ethylenediaminetetraacetate. The enzyme thus obtained was so stable that loss of its activity was undetectable during storage at room temperature for 6 months under air atmosphere, provided that NaCl, KCl or CsCl was present at greater than or equal to 0.7 M.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP. This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography. Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised. As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.

The molecular organization of photochemical reaction (PR) complex in chromatophores from Rhodospirillum rubrum was studied by a combination of proteolytic analysis with proteinase K followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis with rabbit polyclonal antibodies against its five subunits (H, M, L, alpha, and beta). The preparations used for comparison were reaction center complex (RC) (composed of H, M, and L), PR complex, and chromatophores (closed membranous vesicles of polar lipid bilayer having PR complex buried in the membrane). 1. RC was bound with anti-H, anti-M, and anti-L antibodies, whereas PR complex and chromatophores were bound with anti-H and anti-beta antibodies, but not with the other antibodies. 2. With PR complex, H (Mr 31,000 (31K)) was rapidly degraded into two peptides with Mr of 16K and 14.5K (abbreviated as 16K and 14.5K, respectively), M (27K) into 25.5K, and beta (11K) into 10K. Significantly later, the 25.5K of M was degraded into 24K, L (23K) into 19K, and alpha (12K) into 11K. With chromatophores, H and beta were degraded in a manner similar to that with PR complex, whereas M, L, and alpha were not degraded at all. With RC, H, M, and L were rapidly degraded. 3. With RC, the activity for photooxidation of P870 (photochemical activity) was hardly affected till H, M, and L had been degraded into less than 10K, 24K, and 19K, respectively. With PR complex, the absorbance spectrum due to the bacteriochlorophylls of light-harvesting complex-1 composed of alpha and beta (LH1-Bchl) changed in parallel to the degradation of alpha or 10K (a part of beta). 4. Together with the previous results (Ueda et al. (1985) J. Biochem. 98, 1487-1498), the present findings suggest that: 1) RC is directly surrounded by 12 alpha and further by 12 beta; 2) H and beta are mostly and partially exposed, respectively, on the outer surface of the membranous vesicle; 3) a small part of M is exposed on the inner surface of the membranous vesicle.

DNA polymerase A (I or major) and its stimulative factor were purified from 15-20 kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis. The extraction was carried out in the presence of 10 or 3 mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively). These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300. Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME). After ME treatment, however, they showed two protein bands at Mr of about 145,000 and 75,000 in SDS-PAGE, except for those obtained by the procedure involving 10 mM PMSF and the batchwise adsorption-elution. All the preparations described above showed practically the same specific activity. This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145,000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75,000, which were still active and were linked to each other through a disulfide bond. Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20,000 in SDS-PAGE before and after ME treatment. The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38% with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA).oligo(dT)10, respectively. With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated. The purified factor also stimulated the activity of calf thymus DNA polymerase alpha by 150% with denatured salmon sperm DNA; Km was about 5 X 10(-10)M, practically the same as that of yeast DNA polymerase A. However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.