Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

Solubilization and structural stability of bacteriorhodopsin with a mild nonionic detergent, n-Octyl-β-thioglucoside

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.     

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Gl?ckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.     

Isolation and characterization of halorhodopsin from Halobacterium halobium

Chromoprotein of a light-driven chloride pump, halorhodopsin (HR), was isolated from Halobacterium halobium L-33, which contains HR and "slowly cycling rhodopsin-like pigment" (SR) but lacks bacteriorhodopsin (BR). The isolation was run in the presence of more than 2 M NaCl, which was required to preserve this halophilic retinal protein. Cell envelope vesicles were washed with Tween-20 to remove 80% of the proteins. The residual membranes were solubilized with 0.5% C12E9, which had little effect on the photochemical activities of HR and SR. HR was purified by passing it through a hydroxyapatite and then a phenyl-Sepharose column in 2 M NaCl and 0.5% C12E9. The absorption maximum of HR was 578 nm and the ratio of absorbance at 280 nm to 580 nm was 1.52. The apparent molecular weight of HR was 20,000 on polyacrylamide gel electrophoresis in the presence of SDS. The characteristic, bilobed CD spectrum of HR in the visible region suggested that HR exists as an oligomer in both its membrane-bound and isolated forms.     

Photoactive retinal pigments in haloalkaliphilic bacteria

Light-induced fast transient absorbance changes were detected by time-resolved spectroscopy in 38 of 51 haloalkaliphilic isolates from alkaline salt lakes in Kenya and the Wadi Natrun in Egypt. They indicate the presence of two retinal pigments, Pf and Ps, which undergo cyclic photoreactions with half-times of 2 ms and 500 ms respectively. Pf absorbs maximally near 580 nm and Ps near 500 nm. The pigments differ in their sensitivity to hydroxylamine and detergent bleaching and the photoreactions of Pf are strongly dependent on chloride concentration. Of the 38 pigment-containing strains, 29 possess both Pf and Ps, 9 possess only Ps. Inhibition of retinal synthesis with nicotine blocks pigment formation and addition of retinal restores it. Hydroxylamine-bleached pigments can be reconstituted with retinal or retinal analogues. Their similarity to the retinal pigments of Halobacterium halobium strongly suggests that they are also rhodopsin-like retinyledene proteins. Pf in all properties tested is almost identical to halorhodopsin, the light-driven chloride pump of H. halobium, and may serve the same function in the haloalkaliphiles. Ps has photocycle kinetics similar to sensory rhodopsin and a far-blue-shifted long-lived photocycle intermediate, but its ground state absorption maximum is near 500 nm instead of 587 nm. We have not found a bacteriorhodopsin-like pigment in the haloalkaliphiles.     

Adaptation of aerobic methylobacteria to dichloromethane degradation

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCl. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.     

Solubilization and Structural Stability of Bacteriorhodopsin with a Mild Nonionic Detergent,n-Octyl-β-thioglucoside

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.     

In vitro evaluation of the fermentation characteristics of the carbohydrate fractions of hulless barley and other cereals in the gastrointestinal tract of pigs

An in vitro model was used to study the fermentation characteristics of carbohydrate fractions of hulless barley (hB), in comparison to hulled barley (HB), hulled oat and oat groats (OG) in the pig intestine. For this purpose, 6 hulless barley cultivars (hB), varying in β-glucan content (36–99 g/kg DM), were compared to 3 HB cultivars, 2 oat groat samples (OG), 3 oat varieties and a reference sample of wheat. The residue of a pepsin–pancreatin hydrolysis was incubated in a buffered mineral solution inoculated with pig faeces. Gas production, proportional to the amount of fermented carbohydrates, was measured for 48 h and kinetics modelled. The fermented solution was subsequently analyzed for microbial production of short-chain fatty acids (SCFA) and ammonia. In vitro dry matter degradability varied according to ingredient (P<0.001). Higher values were observed for OG, ranging from 0.88 to 0.99 as compared to oat, hB and HB, for which degradability ranged from 0.63 to 0.73, 0.68 to 0.80 and 0.69 to 0.71, respectively. A “cereal type” effect (P<0.05) was observed on fermentation kinetics parameters. Total gas production was higher (P<0.05) with hB (224 ml/g DM incubated) than with HB and oat (188 and 55 ml/g DM incubated, respectively). No difference was observed between hB cultivars (P>0.05) for total gas production but differences (P<0.001) were found for lag time and the fractional rate of degradation. Hulless barley cultivar CDC Fibar (waxy starch) and CDC McGwire (normal starch) started to ferment sooner (lag time of 0.7 and 0.9 h, respectively) than SH99250 (high amylose starch; 1.7 h). The fractional rate of degradation was similar in both hB and OG (0.15/h on average), which was higher than that of HB (0.12/h). The production of SCFA was also higher (P<0.05) with hB (6.1 mmol/g DM incubated, on average) than with HB and oat (4.9 and 2.9 mmol/g DM incubated, respectively). Similar trends were found for SCFA production expressed per g fermented carbohydrates, with higher butyrate and lower acetate ratio. In contrast, oat fermentation generated higher (P<0.05) ammonia concentration (1.4 mmol/g DM incubated, on average) than hB (1.0 mmol/g DM incubated). In summary, hulless barleys, irrespective of cultivar type had higher in vitro fermentability and produced more SCFA and less ammonia than hulled barley and oat. Thus, hulless barleys have a better potential to be used in pig nutrition to manipulate the fermentation activity in the intestine of pigs.     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

In vitro salinity tolerance of two pistachio rootstocks: Pistacia vera L. and P. atlantica Desf

Seedlings of Pistacia vera L. and Pistacia atlantica Desf. were cultured on hormone-free DKW medium supplemented with NaCl. The plants were subjected to low NaCl concentrations ranging from 0 to 80 mM for 45 days or to high salt concentrations (0, 131, and 158.5 mM for P. vera and 0, 131, and 240 mM for P. atlantica) for 25 days. Toxicity symptoms were recorded for seedlings exposed to low NaCl treatments. Plant growth, survival rates, mineral content, as well as proline and soluble sugar contents were determined and evaluated at the end of the culture period. The results indicated that low NaCl treatments yielded no instances of plant death in both species. At high salt conditions, however, significant mortality rates were noted for both species, being 22.86% at 240 mM NaCl for P. atlantica and 25.8% at 158.5 mM NaCl for P. vera. With regards to salinity effects, levels of 60 and 80 mM NaCl induced significant decreases of stem elongation and leaf number in the P. vera species. Salinities between 40 and 80 mM NaCl, however, induced a decrease in the root number of both species. The fresh weights of P. vera and P. atlantica also decreased significantly after 45 days of culture at NaCl concentrations between 40 and 80 mM and after 25 days of culture at 158.5 and 240 mM NaCl, respectively. The sodium and chloride uptake in plant organs seemed to be controlled more efficiently in P. atlantica than in P. vera. In both species, the K⁺ content was noted to undergo a significant decrease when salinity increased. While the K⁺/Na⁺ ratio was maintained above 2 at low NaCl treatments, it was sharply decreased at high NaCl conditions, suggesting a failure of K–Na selectivity mechanism. The Ca²⁺/Na⁺ ratio decreased significantly at 60 and 80 mM NaCl in P. vera and at 60 mM NaCl for P. atlantica. In both Pistacia species, high NaCl treatments (131–240 mM NaCl) induced a significant increase in proline content.     

Synchrotron X-ray scattering study of chromatin condensation induced by monovalent salt: analysis of the small-angle scattering data

Small-angle X-ray scattering experiments were carried out on rat thymus chromatin in "native" and "H1-depleted" states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44 nm, suggesting the structure corresponding to the "10 nm" filament. With increasing NaCl concentration, the Rc value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.     

Degradation of p-nitrophenol by Achromobacter xylosoxidans Ns isolated from wetland sediment

Achromobacter xylosoxidans Ns strain, capable of utilizing p-nitrophenol (PNP) as the sole source of carbon, energy, and nitrogen, was isolated from wetland sediment and confirmed based on 16S rRNA gene sequence. The strain Ns could tolerate concentrations of PNP up to 1.8 mM, and degradation of PNP was achieved in 7 d at 30 °C in the dark under aerobic conditions. Biodegradation of PNP occurred quickly at an optimal pH of 7.0 and higher, and at ⩽0.5% salt (NaCl) contents. During bacterial growth on PNP, 4-nitrocatechol was observed as a key degradation intermediate using a combination of techniques, including HPLC, UV–visible spectra, and comparison with the authentic standard. In a similar way, a second degradation intermediate was identified to be 1,2,4-benzenetriol. Moreover, A. xylosoxidans Ns could also degrade 3-nitrophenol as the sole source of carbon, nitrogen, and energy, but 2-nitrophenol could not. The experimental results showed that bacteria indigenous to the wetland sediment are capable of degradading PNP and chemicals with similar structures.     

Adaptation of aerobic methylobacteria to dichloromethane degradation

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.     

Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.     

Na+- and K+-dependent oligomeric interconversion among alphabeta-protomers, diprotomers and higher oligomers in solubilized Na+/K+-ATPase

Protein fractions of a higher-oligomer (H), (alphabeta)(2)-diprotomer (D) and alphabeta-protomer (P) were separated from dog kidney Na(+)/K(+)-ATPase solubilized in the presence of NaCl and KCl. Na(+)/K(+)-dependent interconversion of the oligomers was analysed using HPLC at 0 degrees C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e. DeltaC(max) = 26.7%. DeltaC(max) for the sum of D and H was equivalent to the absolute value of DeltaC(max) for P, regardless of the anion present, indicating that K(+) induced the conversion of P into D and/or H, and Na(+) had the opposite effect. When enzymes that had been denatured to varying degrees by aging were solubilized, DeltaC(max) increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D <==> 2P, assuming 50-fold difference in the K(d) between KCl and NaCl, and coexistence of unconvertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K(0.5)s of 64.8 microM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na(+)/K(+)-binding to their active transport sites.     

Mechanism of the rate-determining step of the Na(+),K(+)-ATPase pump cycle

The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E(2) conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 +/- 3 s(-1) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 +/- 7 mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.     

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.     

Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase) is active in mammalian pancreas, where it plays a role in the resynthesis of phosphatidylinositol (PI) during agonist-stimulated inositol-phospholipid metabolism. The enzyme was found to be present in relatively high specific activity [30 nmol of PI formed min-1 (mg of protein)-1] in dog pancreas microsomal membranes, and its activity in these membranes was partially characterized. The Km for myo-inositol was 0.76 mM, and the apparent Km for cytidine(5')diphospho-1,2-diacylglycerol (CDP-diacylglycerol) was 18 microM. The apparent Ka values for activation by Mn2+ and Mg2+ were respectively 42 microM and 2.5 mM. The pH optimum was 8.5-9.0. The enzyme was solubilized in stable form and in nearly quantitative yield with 40 mM n-octyl glucopyranoside (OG), with 4-6 mg of OG/mg of microsomal protein. In the presence of solubilizing levels of OG, the enzyme exhibited less than maximal activity, but full activity was restored by dilution of the OG to below its critical micelle concentration of 20-25 mM. The presence of Mn2+ was essential for stabilization of the OG-solubilized enzyme, with half-maximal stabilization at 40 microM Mn2+. The stability of the OG-solubilized enzyme was sufficient to facilitate purification of the enzyme in the presence of this detergent, with 67% of the activity remaining after 3 days at 4 degrees C. The enzyme was partially purified by OG extraction and DEAE-cellulose chromatography, in 98% yield, to a specific activity of 290 nmol of PI formed min-1 (mg of protein)-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments.

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.