Quantization of membrane potential generation by cytochrome c oxidase in small vesicles

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.     

NADPH-specific and NADH-specific xylose reduction is catalyzed by two separate enzymes in Pachysolen tannophilus

Summary Pachysolen tannophilus contains — in addition to an NADPH-linked xylose reductase — a separate NADH-linked one, in this respect differing from the yeast Pichia stipitis. Both enzyme proteins can conveniently be separated from each other by either ion exchange chromatography or chromatofocusing.     

Rationalization of some genetic anticodonic assignments

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization

The major histocompatibility complex (SLA) of the domestic pig (Sus scrofa) was regionally mapped to 7p12----q12 by in situ hybridization with an SLA class I-specific recombinant DNA probe. This localization contradicts linkage data suggesting a possible assignment of the SLA locus to porcine chromosome 15.     

Fine-granular cationic iron colloid. Its preparation, physicochemical characteristics and histochemical use for the detection of ionized anionic groups

In order to obtain distinct and reliable information concerning the localization of ionized anionic groups in tissues, fine-granular cationic ferric hydroxide colloid solution (Fe-Cac-f) was newly devised. This can be obtained by boiling a mixture of ferric chloride and ammonium cacodylate solutions. The colloid particles of Fe-Cac-f are about 1.0 nm in size, i.e., one-fifth of the size of ferric cacodylate colloid (Fe-Cac; Seno et al. 1983a). As with Fe-Cac, Fe-Cac-f particles in the pH range of 1.6-7.6 carry a positive electric charge, but the latter show a better permeation of tissues. Using the Prussian blue reaction, Fe-Cac-f gives a distinct deep-blue color and can be used for the detection of anionic groups of acid mucopolysaccharides and proteins by light microscopy. It is also useful for detecting the exact sites of ionized anionic groups in deep tissue areas using electron microscopy.     

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.