Cytochrome c oxidase. Time dependence of optical and EPR spectral changes related to the ‘oxygen-pulsed’ form

Time-dependent changes in the optical spectrum (450–920 nm) of cytochrome c oxidase, following oxidation with oxygen of the stoichiometrically reduced form, have been investigated and where possible, attempts have been made to correlate our observations with variations in the EPR spectrum over a parallel time course at 2°C. In this regard, particular emphasis has been placed on establishing absorption features related to the presence of EPR resonances at g 5, 1.78 and 1.69, which have been tentatively assigned to a spin-coupled state involving cytochrome a₃ and ‘EPR-undetectable Cu’ (Beinert, H., Shaw, R.W., Dunham, R.W. and Sands, R.H. (1982) in Oxidases and Related Redox Systems (King, T.E., Mason, H.S. and Morrison, M., eds.), Pergamon Press, Oxford, in the press). For optical studies we have used a versatile rapid-scanning spectrophotometer to obtain well resolved spectra down to 2 ms reaction time. Concomitant with the appearance (within 10 ms) of EPR signals at g 5, 1.78 and 1.69 is the presence of an enhanced absorption (Δε = 0.25 mM (heme a)^?1·cm^?1) at 660 nm, with a trough (relative to following spectra) at 580 nm. In our hands, this feature disappears in a first-order process with a half-life of 46 s at pH 7.2 and 2°C. The effect of this spectral transformation is to decrease considerably the acuteness of the 655 nm absorption band, previously suggested as representing a state of the enzyme in which ferric cytochrome a₃ is coupled to oxidised EPR-undetectable Cu (Beinert, H., Hansen, R.E. and Hartzell, C.R. (1976) Biochim. Biophys. Acta 423, 339–355). This observation can be correlated satisfactorily with a small field shift of the high-field resonances at g 1.78 and 1.69 and a broadening at g 1.78. Support for this and further correlative assignments arises from parallel experiments using cytochrome c oxidase purified via an alternative procedure, which displays different kinetic behavior. Further transformations of the oxidized enzyme are evident through an approx. 10% decrease in absorbance at 600 nm together with small changes centered at 640 and 665 nm (which serve to restore the sharpness of the 655 nm band). The kinetics, as analyzed by the Guggenheim procedure using the absorbance at 597 nm, indicate approx. 50% first-order linearity (half-life 40 min) with additional species contributing at longer times, while over a parallel time course (0–3 h) the EPR resonances at g 5, 1.78 and 1.69 virtually disappear. These novel signals can also be seen at a lower intensity in samples of cytochrome c oxidase anaerobically reoxidized by porphyrexide and frozen after a 6 min incubation period at 4°C. This observation, along with the establishment of similar optical changes over the time course of 1 min to 3 h, suggests that aerobic and anaerobic reoxidation produce common forms of the enzyme. Comparison of the g 1.78 and 1.69 resonances between samples rapidly aerobically reoxidized in the presence of H₂¹⁶O and H₂¹⁷O yielded no evidence for the presence of any labile oxygen ligand (including OH^?, H₂O) in the coordination sphere of the species involved.     

The diabetic Zucker fatty rat

A noninsulin-dependent diabetes appeared in fatty rats in our Zucker rat colony. A breeding program yielded a genetic pattern of diabetes consistent with a dominant gene not closely linked to the fatty gene. Fatty males were more frequently affected than fatty females. Since no markers could be identified for heterozygous carriers and since affected fatty rats were 6 months old when diabetes appeared, the diabetic trait could not be sustained in our small colony. Glucose tolerance tests showed that the diabetic fatty rats had little increase in plasma insulin concentration after a glucose load was administered. Plasma insulin concentrations were unchanged relative to control fatty rats. Percentage body fat and plasma triglyceride values were decreased in fatty diabetic rats relative to control fatty rats, however, consistent with insulin resistance in fat and liver. The content of pancreatic insulin was markedly decreased in the diabetic fatty rat relative to either the ad libitum or diet-restricted fatty rats. The occurrence of a genetically based diabetes in a normally outbred colony underscores the importance of genetic traits that interact with obesity in determining diabetes in rodent models.     

Membrane potentials associated with Ca-induced K conductance in human red blood cells: Studies with a fluorescent oxonol dye,WW 781

Summary A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca] _o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on K _o is evident at pH 6, where the ionic selectivity of activation is K, Rb>Cs>Na and that of conductance is K, Rb>Cs. Conditions were defined which permitted continuous monitoring ofE _m for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes inE _m, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects ofE _m on fluorescence.     

Breast reconstruction by superior gluteal microvascular free flaps without silicone implants

The author's experience with 10 gluteus maximus myodermal free flap breast reconstructions is reviewed against the current methods of reconstruction using silicone implants, latissimus dorsi flaps, regional skin flaps, and rectus abdominis myodermal flaps. The superior gluteal free flap can achieve a reliable, permanent, and aesthetic reconstruction of the breast without silicone implants. The softness, projection, natural appearance, and patient satisfaction are excellent compared with other methods. It is particularly useful in patients who object to the use of artificial implants, are not suitable for regional flaps, or have disappointing results from previous reconstructions. Technical modifications of the flap design and selection of the recipient vessels are important.     

Homogeneity of the HLA-Linked SB2 and SB3 specificities demonstrated by cloned alloreactive T cells

The HLA-linked "SB" antigens comprise a new segregant series of B-cell alloantigens mapping between HLA-DR and glyoxylase. They can be detected by secondary proliferative responses of lymphocytes primed against HLA-A, B, C, DR, MB- and MT-compatible stimulators. To asses genetic complexity of the SB-gene region, alloreactive cloned T-cell lines were derived from four reagents detecting specificities designated SB2 and SB3. In two families, products detected by seven different clones segregated with the HLA haplotypes bearing the SB2 or SB3 specificities as recognized by the uncloned reagents. There were no indications that the cloned cells differed from the oligoclonal reagents in their fine specificity. In contrast to previous results with an SB4-associated specificity, in population studies of 25 SB2-positive and 23 SB3-positive donors, no evidence could be found for subtypes of either specificity. Thus, even at the level of recognition by cloned T-cells, both SB2 and SB3 appear to be remarkably homogeneous in the population.     

Structure and expression of cloned murine IFN-alpha genes

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.     

Dissociation by Chelating Agents and Substructure of the Thermophilic Bacteriophage TP84

The thermophilic bacteriophage TP84 is dissociated into its head, tail, and released deoxyribonucleic acid (DNA) by chelating agents such as ethylenediaminetetraacetic acid (EDTA) and phosphate. The phage is more sensitive to EDTA than to phosphate, and dialysis against either agent causes more effective dissociation than standing in their presence. The tail possesses a knobbed structure which is inserted into the head of the intact phage and to which the DNA appears to be attached. The method of dissociating TP84 described in this paper provides a source of undamaged structural components and intact strands of DNA for subsequent investigations. A possible mechanism of chelate inactivation is discussed.     

Comparative Zone Electrophoresis of Enzymes of Pseudomonas solanacearum and Pseudomonas cepacia

The technique of starch-gel electrophoresis with specific staining for a series of enzymes was used to compare 21 Pseudomonas strains representing both P. cepacia and P. solanacearum. These experiments produced no evidence for close similarity of the two species. Twelve strains of P. solanacearum were compared by means of data obtained from nine different enzymes, and the data indicate that these strains belong in two biotypes. Except for the assignment of two strains, these groups are the same as the two major groups previously derived from nutritional properties and from deoxyribonucleic acid hybridization experiments. Eleven enzymes were available for comparisons of the P. cepacia strains. Eight of these strains form a homogeneous group, but the last strain, number 249, differs considerably from the other representatives of the species.     

Altered proteins with triosephosphate isomerase activity in suppressor-containing strains of Bacillus subtilis

Suppressor mutations in Bacillus subtilis cause the synthesis of a new protein with the enzymatic activity of l-leucine dehydrogenase and two groups of new proteins with the activity of triosephosphate isomerase. The new isoenzymes of triosephosphate isomerase are separable by zone electrophoresis and differ among themselves in elution behavior upon gel permeation chromatography. One group has an apparent average molecular weight of 120,000 to 135,000, which is more than twice that of the wild-type enzyme. Another group appears to be even higher in molecular weight. These data are consistent with the working hypothesis that the new isoenzymes are produced by extension of growing polypeptide chains through one or more chain-terminating triplets, although other mechanisms resulting in alteration of shapes, charges, or associations of the enzymes are not excluded.