Biomedical subjects

M A de la Rosa

Publications and source records attributed to M A de la Rosa.

6 recordsLinked to original sources

PubMed1995

Site-specific mutagenesis demonstrates that the structural requirements for efficient electron transfer in Anabaena ferredoxin and flavodoxin are highly dependent on the reaction partner: kinetic studies with photosystem I, ferredoxin:NADP+ reductase, and cytochrome c.

Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-flow spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been shown to result in very large inhibitions of electron transfer to FNR, were found to yield wild-type behavior in reactions with PSI and cytc. In general, the effects of Fd mutagenesis on the PSI reactions were considerably smaller than those observed for the FNR reaction. In the case of Fld, mutagenesis was found to have only small effects on both the FNR and PSI reactions, although the specific sites whose mutation caused changes in electron transfer properties differed for the two systems. In contrast, several of the Fld mutants showed appreciably larger effects on the nonphysiological reaction with cytc. We conclude from these studies that the structural requirements for efficient electron transfer involving the Fd and Fld molecules differ, depending upon the reactant with which these redox proteins interact. This is consistent with the multiple roles that these proteins have in vivo in biological electron transfer and implies that different conserved residues in these proteins have evolved to satisfy varying requirements of particular reaction partners.

Anabaena↗

PubMed1993

A comparative laser-flash absorption spectroscopy study of Anabaena PCC 7119 plastocyanin and cytochrome c6 photooxidation by photosystem I particles.

Laser-flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c6 and plastocyanin, isolated from Anabaena PCC 7119, to oxidized P700 in photosystem-I particles isolated from the same cyanobacterium and from spinach. For all metalloproteins and photosystems, the observed rate constant has a non-linear protein-concentration dependence, thus suggesting complex formation preceding electron transfer. Plastocyanin and cytochrome c6 have similar association constants for complex formation with spinach photosystem I, but the copper protein exhibits a higher intracomplex-electron-transfer rate constant (twofold). With Anabaena photosystem I, the two redox proteins are more effective with respect to both complex formation (5-10-fold) and electron transfer (1.5-4-fold) than with the spinach photosystem. In all cases, the observed rate constants for electron-transfer monotonically decrease with increasing NaCl or MgCl2 concentration. This is interpreted in terms of the involvement of attractive electrostatic interactions, which result in the initial collision complex having the most productive orientation for the electron transfer process, without a requirement for further reorientation. The magnitude of the response to MgCl2 suggests the occurrence of specific ion effects as well. In the absence of added salts, the reduction rate of oxidized P700 increases with pH from approximately 6 to 8, but decreases slightly at pH 8.5.

Anabaena↗

PubMed1992

Further development of the baboon as a model for acute schistosomiasis.

Baboons develop a syndrome, including eosinophilia and transient fever, after infection with cercariae of Schistosoma mansoni that is consistent with the human syndrome of acute schistosomiasis. Radiotelemetry can be used to follow the course of fever in infected baboons. Individual variations in intensity of disease were noted in baboons. These symptoms and signs were more closely linked to the onset of oviposition by the newly matured worms than they were to the presence of migrating schistosomula or maturing worms. The baboon is concluded to be a suitable and useful model for human acute schistosomiasis mansoni.

Acute Disease↗

PubMed1984

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria.

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.

Acid-Base Equilibrium↗

PubMed1980

Purification and properties of assimilatory nitrate reductase [NAD(P)H] from Ankistrodesmus braunii.

Assimilatory nitrate reductase [NAD(P)H] (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by a simple procedure that utilizes as the main step affinity chromatography on Blue-Sepharose. The best enzyme preparation has a specific activity of 61.25 units/mg protein. The enzyme has a sedimentation coefficient of 10.9 S by sucrose-density-gradient centrifugation, and a Stokes radius of 9.8 nm was estimated by gel filtration techniques. Its molecular weight is 460000, but only one single band of 58000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of eight subunits. The nitrate reductase absorption spectrum shows wavelengths maxima at 280 and 416 nm and a broad shoulder at 450 nm. Reduced enzyme shows maxima at 424 (Soret), 527 (beta) and 557 (alpha) nm, and a bleaching at 450 nm. The reduced extracted heme chromophore, in pyridine and KOH, shows absorption bands at 414, 522 and 552 nm. These properties indicate the presence of a b-type cytochrome and flavin as prosthetic groups of A. braunii nitrate reductase. A minimum of four molecules of heme has been calculated per molecule of the enzyme complex. Redox titration of the enzyme shows a midpoint potential for the heme of -73 mV at pH 7.0. In the presence of p-hydroxymercuribenzoate, which inhibits the NAD(P)H-dependent activities of the complex, the enzyme-bound heme can be reduced with dithionite, but not with NAD(P)H.

Centrifugation, Density Gradient↗

PubMed1980

[Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)].

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.

Chlorophyta↗