Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Tyrosine aminotransferase deficiency in mink (Mustela vision): A model for human tyrosinemia II

Mink pseudodistemper, a recessive disease associated with high blood tyrosinelevels, is an animal analogue of the human inborn error of metabolism, tyrosinemia II. Affected mink and man have eye and skin lesions. Affected mink have no hepatic tyrosine aminotransferase (TAT) activity, as measured immunologically and biochemically. Hepatic mitochondrial aspartate aminotransferase is increased to 188% of control. This new genetic animal model of TAT deficiency should allow new studies of tyrosine metabolism.Supported by NIH Grants AM-17253 (LAG), 5T32-AM-07093 (LAG), and RCDA AM-0008 (LAG), grants from the Wisconsin State Mink Advisory Board, and a BRSG grant for the Graduate School of the University of Wisconsin, Madison, Publication No. 82 of the dermatology research laboratories of Duke University Medical Center.     

Protein--immobilized lipid in dimyristoylphosphatidylcholine-substituted cytochrome oxidase: evidence for both boundary and trapped-bilayer lipid.

Cytochrome oxidase-dimyristoyl phosphatidylcholine complexes have been prepared at defined lipid:protein ratios to study the effects of protein packing density on the lipid fluidity. All the complexes reveal a two-component ESR spectrum from an incorporated phosphatidylcholine spin label, corresponding to both an immobilized lipid boundary layer and fluid bilayer regions. Difference spectra, obtained by subtracting the same immobilized spectrum from the spectra of the various complexes, demonstrate a strong perturbation of the lipid bilayer fluidity which is quite distinct from the immobilized boundary layer formation.     

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.     

A spin-label study of the chromaffin granule membrane.

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied. 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32-36 degrees C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 degrees C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature. 3. The lipid-soluble spin label 2,2,6,6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 degrees C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possible involving a re-distribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105-114(1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.     

Interrelationships between prostaglandins, cyclic AMP and steroids in ovulation.

Ovulation is a complex phenomenon, involving a series of biochemical events within the ovary, leading to the rupture of the follicle. This paper summarizes recent studies in our laboratory of some of these biochemical changes using the rabbit as an experimental model. It has been shown in our laboratory that isolated Graafian follicles obtained from oestrous rabbits synthesize steroids and cyclic AMP when incubated in vitro. Luteinizing hormone added to the incubation medium increased steroidogenesis and cyclic AMP synthesis many fold. When follicles were isolated from rabbits at different times following the ovulatory stimulus (mating or HCG injection) it was found that the in vitro response to LH in terms of steroidogenesis and cylcic AMP synthesis was lost as ovulation approached. In contrast, when prostaglandins (PGF and PGE) were measured in rabbit Graafian follicles it was found that the PGF and PGE levels increased as ovulation approached. From these data and from reports in the literature, we have developed a hypothetical model for ovulation in the rabbit which may help in a better understanding of the ovulatory process.