Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS.

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Delta32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.     

Myeloperoxidase catalysed cooxidative metabolism of methimazole: oxidation of glutathione and NADH by free radical intermediates

The myeloperoxidase catalysed oxidation of methimazole in the presence of NADH or GSH resulted in oxygen uptake suggesting that metabolism proceeded via a one electron mechanism. The GSH was oxidised to GSSG and the thiyl radical could be trapped with DMPO while NADH was oxidized to NAD+. Metabolism proceeded without the inactivation of the enzyme myeloperoxidase. Myeloperoxidase catalyzed oxidation of other substrates which proceed via one electron intermediates; 2,6-dimethylphenol, N,N,N',N'-tetramethyl-phenylenediamine and luminol, were all stimulated by methimazole providing further evidence for a methimazole free radical. The presence of iodide stimulated the oxidation of methimazole but inhibited the oxygen uptake in the presence of GSH or NADH suggesting that metabolism in this case proceeded by a two electron mechanism. In contrast, another S-thioureylene drug, thiourea; did not cause oxygen uptake when oxidised in the presence of GSH or NADH indicating that the myeloperoxidase oxidation of thiourea proceeded primarily by a two electron mechanism. The horseradish peroxidase catalysed one electron oxidation of p'p'-biphenol, and 3,3',5,5'-tetramethylbenzidine was reversibly inhibited by methimazole and thiourea by preventing the accumulation of oxidation products via reductive mechanisms whereas the reversible inhibition of guaiacol and luminol oxidation was the result of competitive inhibition. With p,p'-biphenol, and 3,3',5,5'-tetramethylbenzidine unstable adduct formation could be demonstrated.     

Influence of albumin and non-esterified fatty acids on serum prostacyclin binding in pregnancy

We investigated the etiology of the previously documented decrease in serum prostocyclin binding during pregnancy. Addition of albumin to the serum of pregnant women failed to raise binding to non-pregnant levels. Pregnancy serum bound significantly more prostacyclin following the removal of non-esterified fatty acids and the addition of fatty acid free albumin resulted in a rise in binding to non-pregnant levels. We conclude that serum protein prostacyclin binding is affected by both albumin concentration and non-esterified fatty acids.     

The α-glycerophosphate cycle inDrosophila melanogaster. I. Biochemical and developmental aspects

The two α-glycerophosphate dehydrogenases ofDrosophila melanogaster (mitochondrial αGPO and soluble αGPDH) have been biochemically characterized in a preliminary investigation of the α-glycerophosphate cycle inDrosophila. The soluble enzyme is NAD linked and can be distinguished from the mitochondrial oxidase in terms of locational specificity,pH optimum, salt precipitation, and electrophoretic behavior. The mitochondrial enzyme is NAD independent and exhibits behavior typical of a lipoprotein. Extraction procedures are described for αGPO with nonionic detergents. Isoelectric focusing of αGPO on polyacrylamide gels resolved two molecular forms of αGPO which differ in isoelectric point, ease of extraction, and developmental and spatial distribution. Developmental profiles of both αGPO and αGPDH are presented. The occurrence of multiple forms of both the soluble (Wright and Shaw, 1969) and the mitochondrial forms of the enzymes is discussed in light of a multifunctional role of the α-glycerophosphate cycle inDrosophila. This project was supported by a Genetics Training Grant T1 GM 1035 from the National Institute of General Medical Sciences.