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H Wohlrab

Publications and source records attributed to H Wohlrab.

At least 37 records · Page 2Linked to original sources
PubMed1985

Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria.

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.

Amino Acid Sequence↗
PubMed1985

Reversed-phase high-performance liquid chromatographic separation and quantitation of phenylthiohydantoin derivatives of 25 amino acids, including those of cysteic acid, 4-hydroxyproline, methionine sulfone, S-carboxymethylcysteine and S-methylcysteine.

A high-performance liquid chromatography system is presented which allows separation and quantitation (in the range 4-1000 pmol) of all common phenylthiohydantoin amino acids, including derivatives of 4-hydroxyproline, methionine sulfone and three differently modified forms of cysteine. By showing the actual solvent gradient during elution (as opposed to the programmed gradient) and by supplying information on the effects of minor changes in solvent-pH, column temperature, flow-rate, and concentration of 2-propanol in the gradient, we make guidelines available for fine-tuning the separation with new Ultrasphere-ODS (C18) columns.

Amino Acids↗
PubMed1984

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.

Amino Acids↗
PubMed1982

pH gradient-dependent phosphate transport catalyzed by the purified mitochondrial phosphate transport protein.

The mitochondrial phosphate transport protein (Wohlrab, H. (1980) J. Biol. Chem. 255, 8170-8173), which co-purifies with the adenine nucleotide translocase, has been isolated with a modified procedure resulting in an increased yield and in a preparation that is stable to storage in liquid nitrogen. The phosphate transport protein was incorporated into liposomes (phosphatidylethanolamine/phosphatidylcholine/phosphatidic acid, 2.75:1:1), and phosphate/phosphate exchange rates were determined. With pHe (extraliposomal) = pHi (intraliposomal) = 7.2, we found a Km of 2.5 mM independent of [(Pi)i] and a Vmax of 12 mumol/min . mg of protein. Parallel phosphate transport experiments were carried out with liposomes containing phosphate transport protein isolated from mitochondria inhibited by N-ethylmaleimide. Phosphate transported unidirectionally [pHe = pHi = 6.8; = 1.0 mM, (Pi)i = 0 mM] reaches a maximum at 30 s and is 0.13 and 0.05 mumol/mg for active and inhibited protein, respectively. At pHe = 6.8 and pHi = 8.0, the respective amounts are 0.45 and 0.05. At pHe = 8.0, the uptake becomes the same with active and inhibited protein (pHi = 6.8, 0.15 and 0.14; pHi = 8.0, 0.25 and 0.20). At all the above pH values, about the same uptake is observed with liposomes prepared without protein as those prepared with inhibited protein. The initial rate of protein catalyzed unidirectional flux [(Pi)e = 1.0 mM, pHe = 6.8; (Pi)i = 0 mM, pHi = 8.0) is 2 mumol/min . mg of protein or 8 mumol/min . mg of phosphate transport protein estimated without the adenine nucleotide translocase.

Animals↗
PubMed1979

Identification of the N-ethylmaleimide reactive protein of the mitochondrial phosphate transporter.

The mitochondrial phosphate carrier is inhibited by the SH reagents p-(hydroxymercuri)benzoate and N-ethylmaleimide. Based on an analysis utilizing dodecyl sulfate-polyacrylamide gels, an SH-containing 32 000-dalton protein has been identified as a component of the phosphate carrier system. Two other N-[3H]ethylmaleimide-labeled proteins of the inner mitochondrial membrane have been eliminated from this role [Wholrab, H., & Greaney, J., Jr. (1978) Biochim. Biophys. Acta 503, 425] on the basis that band IV (45,000 daltons) is absent from heart sonic submitochondrial particles and band VII (6 500 daltons) does not react with p-(hydroxymercuri)benzoate. The mobility of the 32 000-dalton protein (0.43) is lower than that of the gamma subunit of the mitochondrial ATPase (0.46) and the carboxyatractyloside binding protein (0.48) on 12.5% dodecyl sulfate-polyacrylamide gels. In these flight muscle mitochondria, 0.87 nmol of N-[3H]ethylmaleimide per nmol of cytochrome a is bound to the 32,000-dalton protein.

Animals↗
PubMed1978

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane.

Mitochondria have been prepared from the flight muscles of mature blow-flies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome A inhibit the swelling by 90%. When the mitochondria are inhibited by N-[3H]ethylmaleimide, then solubilized in dodecyl sulfate/mercaptoethanol at 100 degrees C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the N-[3H]ethylmaleimide-labeled mitochondria, solubilized, and electrophoresed on dodecyl sulfate-polyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 . 10(3). The nmol N-[3H]ethylmaleimide bound to each of these proteins per nmol cytochrome A are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol N-[3H]ethylmaleimide/mol cytochrome a at 10 micron cytochrome A. Coty and Pedersen (1975) J. Biol. Chem. 250, 3515-3521) sensitized rat liver mitochondria to N-[3H]ethylmaleimide and identified five labeled proteins. Only the labeled 32 . 10(3) dalton and the 45 . 10(3) dalton proteins are common to both systems.

Animals↗
PubMed1976

Age-related changes in the flight muscle mitochondria from the blowfly Sarcophaga bullata.

Flight muscle mitochondria have been isolated from female blowflies (Sarcophaga bullata) of different ages, alpha-Glycerophosphate and pyruvate-proline respiration rates increase during development. Only pyruvate-proline respiration declines toward senescence (30%). This decline can be overcome by ATP-NaHCO3. Cytochrome concentrations and hydrogen peroxide generation rates per protein increase during development but remain constant thereafter. Total NAD+ of metabolically completely oxidized mitochondria decreases during development; a small decline occurs between mature and senescent mitochondria. Respiring young mitochondria do not swell in potassium isethionate, very little in potassium chloride, and relatively slowly in potassium acetate. Mature and senescent mitochondria do swell in these three salts but cannot be differentiated from each other on this basis. None of the preparations swells in sodium chloride, sodium- or potassium Mops. While many differences exist between young and mature mitochondria, only the decline in pyruvate-proline respiration distinguishes mature from senescent mitochondria.

Aging↗