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Biomedical subjects

J B Jackson

Publications and source records attributed to J B Jackson.

At least 127 records · Page 7Linked to original sources
PubMed1991

Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus.

1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.

Bacterial Chromatophores↗
PubMed1991

Purification and properties of dimethyl sulphoxide reductase from Rhodobacter capsulatus. A periplasmic molybdoenzyme.

Dimethyl sulphoxide reductase was purified from the photosynthetic bacterium Rhodobacter capsulatus. The enzyme is composed of a single polypeptide of Mr 82,000 and contains a pterin-type molybdenum cofactor as the only detectable prosthetic group. The oxidized molybdenum cofactor of dimethyl sulphoxide reductase is a weak chromophore and exhibits broad absorption bands in the u.v.-visible-absorption spectral region. A distinct spectrum was generated upon addition of dithionite.

Chromatography, Gel↗
PubMed1991

The proton-translocating nicotinamide adenine dinucleotide transhydrogenase.

H(+)-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H). There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria and Escherichia coli. Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme. When delta p = 0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting. An increase in delta p accelerates the rate of interconversion in the direction of NADH----NADP+ until another kinetic component, possibly product release, becomes limiting. The available data are consistent with either direct or indirect mechanisms of energy coupling.

Animals↗
PubMed1991

Antibodies to hepatitis C virus in autologous blood donors.

Hepatitis C virus (HCV) is the major cause of posttransfusion hepatitis. Two anti-HCV enzyme immunoassay (EIA) kits and one recombinant immunoblot assay (RIBA) were used to test serum samples of 1476 donations from 692 autologous blood donors to assess the prevalence of anti-HCV and its relationship to transfusion history. Of all autologous blood donations, 23 (1.6%) reacted when tested with one EIA kit and 29 (2.0%) reacted when tested by the other EIA kit. Of the autologous donors, 12 (1.78%) reacted by the first EIA kit and 14 (2.02%) by the second. Discrepancies in the EIA results from different donations by the same donor were seen in seven donors. The RIBA was positive or indeterminate in 33 percent of the EIA-reactive donations and in 41 percent of EIA-reactive donors. All RIBA-positive and -indeterminate samples reacted with both EIA kits. There was no significant difference in the EIA-reactive rates of autologous and first-time homologous blood donors. Previously transfused autologous blood donors had a higher anti-HCV EIA-reactive rate than nontransfused autologous donors, but the difference was not significant. In regard to hepatitis C, the use of autologous blood for homologous transfusion appears to be as safe as the use of blood from first-time homologous donors. Universal testing of previously transfused patients for hepatitis C appears premature at this time. Discrepant anti-HCV EIA results from different donations from the same individual have implications regarding donor deferral.

Adolescent↗
PubMed1991

Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants.

Two non-isotopic polymerase chain reaction (PCR) methods were evaluated by testing blood from 41 HIV-1-seropositive and 16 HIV-1-seronegative Ugandan mothers and 56 of their children (aged 0.5-15.0 months). Amplification of HIV-1 sequences was performed in duplicate using a biotinylated primer pair to the gag region (SK 462-431) and nested primer pairs (JA 17-20) to the pol region of HIV-1. gag sequences were hybridized using a microtiter plate coated with the SK 102 probe followed by colorimetric detection using an avidin-horseradish peroxidase conjugate and tetramethylbenzidine/peroxide substrate. pol sequences were detected on agarose gel stained with ethidium bromide. Results of HIV-1 PCR analysis showed that 40 out of 41 (98%) seropositive mothers and 10 out of 29 (34%) seropositive children had detectable HIV-1 gag and pol sequences. None of the 16 seronegative mothers nor 27 seronegative or Western blot-indeterminate children had detectable HIV-1 sequences. Our results suggest that non-isotopic PCR methods are sensitive, specific, and potentially useful in the early diagnosis of HIV-1 infection in developed and developing countries.

Adolescent↗
PubMed1990

A rapid burst preceding the steady-state rate of H(+)-transhydrogenase during illumination of chromatophores of Rhodobacter capsulatus. Implications for the mechanism of interaction between protonmotive force and enzyme.

At the onset of illumination of chromatophores there was a burst (t1/2 approx. 5 ms) in the rate of the H(+)-transhydrogenase reaction before establishment of the steady-state rate. The burst was suppressed at high pH with a pKa of approx. 8.5. The burst and the steady-state rate were inhibited by either (i) a combination of myxothiazol and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, or (ii) NAD+, or (iii) dicyclohexylcarbodiimide. The results support a model in which substrate binding to H(+)-transhydrogenase is relatively fast. A subsequent slow step is accelerated by the protonmotive force and a third step, possibly product release, is rate-limiting in steady-state turnover during illumination.

Hydrogen-Ion Concentration↗
PubMed1990

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex.

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.

Cell Fractionation↗
PubMed1990

The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus.

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.

Cytochrome c Group↗
PubMed1990

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+.

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia↗
PubMed1990

Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV-1.

To determine whether apparently healthy persons who have had repeatedly reactive enzyme immunoassays and an indeterminate Western blot assay for antibody to the human immunodeficiency virus type 1 (HIV-1) are infected with HIV-1 or HIV-2, we studied 99 such volunteer blood donors in a low-risk area of the country. The subjects were interviewed about HIV risk factors. Coded blood specimens were tested again for HIV-1 antibody (by two different enzyme immunoassays, a Western blot assay and a radioimmunoprecipitation assay) and for HIV-2 antibody by enzyme immunoassay, for HIV-1 by the serum antigen test, for HIV-1 by culture, for human T-cell leukemia virus Type I or II antibody by enzyme immunoassay, and for sequences of HIV DNA by the polymerase chain reaction. Of the 99 blood donors, 98 reported no risk factors for HIV-1 infection; 1 donor had used intravenous drugs. After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66 percent) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92 percent) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2.

Adult↗
PubMed1990

The polymerase chain reaction in transfusion medicine.

PCR is a sensitive and powerful diagnostic and research tool. Its diagnostic applications in infectious disease, genetics, and cancer will play a prominent role in transfusion medicine in the next several years. However, the indirect effect of PCR on transfusion medicine in terms of increasing our understanding of disease mechanisms and the development of therapeutic agents will be even greater.

Acquired Immunodeficiency Syndrome↗