Properties of 5′-nucleotidase in rat heart sarcolemma

The activity of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5′-nucleotidase had a high affinity for AMP (K_m 35 μM), and ATP was a potent competitive inhibitor. In contrast, the 5′-nucleotidase displayed by fraction S showed a low substrate affinity (K_m 130 μM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5′-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5′-nucleotidase at 200 μM relative to 50 μM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5′-nucleotidase activity in both membrane preparations at a concentration of 2 μM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5′-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5′-nucleotidase are present at the intra and extracellular surface of the rat heart sarcolemma.     

Distribution of cholesterol between the outer and inner halves of the lipid bilayer of mycoplasma cell membranes

Rapid kinetic studies of filipin binding to intact cells and isolated membranes were performed with a stopped-flow apparatus to determine the distribution of cholesterol in the outer and inner surfaces of mycoplasma membranes. The initial rates of association of filipin with cholesterol in Mycoplasma gallisepticum and Mycoplasma capricolum intact cells were slower than those obtained with isolated membrane preparations. Ratios of the second-order rate constants for filipin binding to cells relative to membranes indicate that cholesterol is distributed symmetrically in membranes of M. gallisepticum cells whereas in M. capricolum ～66% of the free cholesterol is localized in the outer half of the lipid bilayer.     

Purification of plasma membranes of rat mammary gland : Comparisons of subfractions with rat milk fat globule membrane

Partially purified plasma membranes of rat mammary gland, obtained as light (F₁) and heavy (F₂) fractions by flotation on a discontinuous sucrose density gradient, were further fractionated by density perturbation flotation using digitonin to shift the density of the cholesterol-rich portion of the membranes. The shifted fraction (F₁F₃) of digitonin-treated F₁ was highly enriched in 5′-nucleotidase, cholesterol and sialic acid, but free of galactosyltransferase, suggesting that it contained highly purified plasma membranes. The unshifted fraction (F₁DF₁) was enriched in galactosyltransferase and depleted in nucleotidase, cholesterol and sialic acid, suggesting that it contained Golgi fragments. The F₂ fraction shows substantially different behavior. Part of it re-equilibrates to the F₁ position upon reflotation. When treated with digitonin, part of F₂ is shifted to a higher density (F₂DF₃). F₂DF₃ is enriched in 5′-nucleotidase, cholesterol, sialic acid and galactosyltransferase. These properties suggest that this subfraction comes from a plasma membrane containing galactosyltransferase.The sialoglycoproteins of the various fractions were compared with those of rat milk fat globule membrane, which is derived in part from the apical surface of the mammary secretory cell. Dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals two major glycoprotein bands (GP-II and GP-III) in F₁DF₃. F₂DF₃ contains these and an additional band of lower mobility (GP-I). Both crude and purified MFGM contain all three bands. Comparisons of peanut lectin receptors by autoradiography of polyacrylamide gels run in SDS and then treated with [¹²⁵I]peanut lectin also suggest that F₂DF₃ is more similar to the milk fat globule membrane than is F₁DF₃. However, analysis of the membrane polypeptides and concanavalin A (ConA) receptors shows no obvious relationship between milk fat globule membrane and any of the isolated mammary membrane fractions. These results indicate that the relationship between the milk fat globule membrane and mammary membranes is complex, possibly involving components not associated with the mammary plasma membrane or only selected components of the plasma membrane.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000×g, followed by 90,000×g and finally at 150,000×g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000×g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000×g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO₂ fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N′-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30–65% inactivation over a concentration range of 5–50 μM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5·10⁵ M^?1·min^?1. The correlation between the initial binding of [¹⁴C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F₀F₁-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [¹⁴C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K⁺-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F₀F₁-ATPase complex.     

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 – a 2H NMR study

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 μm α-deuterated palmitic acid (16:0-d ₂) and 75 μm α-deuterated oleic acid (18:1c-d ₂), or with 150 μm 18:1c-d ₂. The fatty acids were incorporated into the membrane lipids and ²H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (H_II) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an H_II phase below 70 °C. The H_II phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (L _α ) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (T _LH) for the L _α to H_II phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the T _LH value. Physiologically relevant concentrations of Ca²⁺ and Mg²⁺ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall. Received: 10 March 1997 / Accepted: 4 July 1997     

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1′-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of ‘marker’ enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C₁₆ alkenyl groups were hydrolysed more rapidly than C₁₈ alkenyl groups. With ³H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C₁₄C₁₈ aldehydes (K_m, 2 μM).     

Isolation and Characterization of a Novel Antialgal Allelochemical from Phragmites communis

Antialgal allelochemicals were isolated from Phragmites communis Tris. The isolated allelopathic fraction showed strong inhibition activity on the growth of Chlorella pyrenoidosa and Microcystis aeruginosa but had no inhibition on Chlorella vulgaris. The 50% effective concentrations (EC₅₀) of the allelopathic fractions on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.79 mg/liter, respectively. The allelopathic activity of the fraction was species-specific. The isolated allelopathic fraction caused metal ion leakage from algal cells. The fraction decreased the activities of antioxidant enzymes, such as superoxide dismutase and peroxidase. The addition of the isolated fraction increased the concentration of unsaturated lipid fatty acids in cell membrane of C. pyrenoidosa and M. aeruginosa. This caused a change in plasma membrane integrity and the leakage of ions in the protoplast. The allelopathic compound was identified by nuclear magnetic resonance and gas chromatography-mass spectrometry as ethyl 2-methylacetoacetate. Synthesized ethyl 2-methylacetoacetate also showed allelopathic activity on C. pyrenoidosa and M. aeruginosa. The EC₅₀ of synthesized ethyl 2-methylacetoacetate on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.65 mg/liter, respectively.     

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-β-cyclodextrin. Role of low-density caveolae-like membranes

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5′nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-β-cyclodextrin (MβCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.     

Chromatographic Characterization and Phytotoxic Activity of Fusarium oxysporum f. sp. albedinis and Saprophytic Strain Toxins

The bayoud disease, vascular fusariosis of date palm tree (Phoenix dactylifera L.), is caused by the pathogenic fungus Fusarium oxysporum f. sp. albedinis. The characteristic symptoms of the bayoud disease were elicited on detached leaves of F. oxysporum f. sp. albedinis‐susceptible cultivars of date palm trees, which were treated either with the F_II (F. oxysporum f. sp. albedinis) fraction purified from the organic extracts of a F. oxysporum f. sp. albedinis liquid culture, or with a solution of fusaric acid. Enniatins, which are secreted by several Fusarium species, were tested at different concentrations and were not capable of inducing symptoms on such detached leaves. The F_II (F. oxysporum f. sp. albedinis) fraction was unable to induce necrosis of potato slices, which indicates that it does not contain significant amounts of enniatins. The high‐performance liquid chromatography (HPLC) profiles of the F_II (F. oxysporum f. sp. albedinis) fraction showed toxic peaks different from fusaric acid. A fraction, named F_II (AZ₄), was obtained from culture filtrates of a saprophytic Fusarium strain maintained in the same cultural conditions as for the F. oxysporum f. sp. albedinis. The HPLC profile of the F_II (AZ₄) fraction did not show the characteristic phytotoxic peaks present in the F_II (F. oxysporum f. sp. albedinis) fraction. This finding well agrees with the fact that the F_II (AZ₄) fraction is not toxic to detached date palm leaves. Moreover, the HPLC profiles of F_II fractions obtained from other special forms of F. oxysporum are different the F_II (F. oxysporum f. sp. albedinis) profile. The phytotoxic compounds purified from the F_II (F. oxysporum f. sp. albedinis) fraction are probably new molecules that may help in understanding the pathogenesis of bayoud disease.     

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from Photosystem II

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F_v/F_m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G _M1 ³ species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to G_M2, G_M1, G_D1a, G_D1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that G_M1 is the major ganglioside species available for enzyme oxidation at the surface.     

Immunological Analysis of Mycoplasma Membranes

The antigens responsible for the production of antibodies to Mycoplasma laidlawii and M. gallisepticum causing growth and metabolic inhibition of these organisms were localized in the cell membrane. Various membrane fractions were tested for serological activity. Membrane lipids were completely or almost completely inactive, whereas several preparations of defatted membrane proteins retained some serological activity, shown by their ability to stimulate metabolic inhibition antibody in rabbits and to adsorb metabolic inhibition antibody and form precipitation lines with an antiserum to the membrane. When the membranes were heated to 65 C for 1 hr, they virtually lost their ability to adsorb metabolic inhibition antibody, which suggests that the antigenic determinants are proteins. Serological activity was retained in reaggregated membranes obtained by dialysis against Mg²⁺ of membranes solubilized in sodium dodecyl sulfate. The amount of solubilized membrane protein and lipid incorporated into the reaggregated membranes could be regulated by varying the Mg²⁺ concentration. As the serological tests indicated that the various membrane antigens were selectively incorporated into the different reaggregated membranes, the use of controlled reaggregation of solubilized membranes is suggested as a new tool for the fractionation and antigenic analysis of membrane proteins.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

Hydrogen oxidation activity in membranes from Rhizobium japonicum

Membranes capable of oxidizing H₂ with O₂ as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H₂-uptake specific activities when isolated in the presence of an H₂ atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O₂-dependent H₂-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H₂-uptake activity was sedimented. The particulate and soluble fractions containing H₂-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H₂ uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H₂ uptake with O₂ as acceptor in membranes was approx. 7.2. H₂-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H₂-reduced minus O₂-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H₂-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H₂-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H₂-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.