Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Potentiation by calcium ions of the antithrombin III inhibition of thrombin

Calcium ions potentiated heparin-modulated antithrombin III inhibition of amidolysis catalysed by thrombin. Potentiation by calcium ions of heparin-independent antithrombin III inhibition of thrombin activity appeared to contribute to this effect. These results suggest a complex modulatory role for calcium ions in proteinase-catalysed reactions influenced by anti-proteinases and glycosaminoglycans.     

Simulation of fermentation conditions for vitamin B12 biosynthesis from whey

Vitamin B₁₂ production in fermentation of Propionibacterium shermanii and Propionibacterium arl AKU 1251 in whey permeate medium has been studied. The observed results and simulated expected values obtained by fitting statistical equations to the recorded data showed that 24 h old inoculum, 5 mg iron l^?1 and 4% whey lactose were optimal for vitamin B₁₂ biosynthesis in both strains when fermentation was carried out under anerobic (84 h) and aerobic (84 h) conditions at 30°C. The supplementation of whey medium with 0.5% (NH₄)₂HPO₄ enhanced further the metabolite yield; however, the preference for a mixed carbon source (lactose + d-glucose or lactose + d-fructose) at different levels varied in the strains under study. P. shermanii, under optimal cultural conditions, was found to be a better strain than Propionibacterium arl AKU, 1251 in fermenting whey lactose for product (vitamin B₁₂) formation.     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis

Characterization of a $\text{Salmonella}\text{typhmurium}$ mutant strain (TA98/1,8-DNP₆) resistant to the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitroarenes) revealed that it was also non-responsive to the mutagenic action of nitroso- and N-hydroxylaminoarenes. The mutant strain was fully sensitive to the mutagenic action of the corresponding hydroxamic acid ester. These results suggest that TA98/1,8-DNP₆ is deficient in a specific esterifying enzyme and that esterification of the penultimate mutagenic metabolites of nitro- and aminoarenes ($\text{e.g.}$, arylhydroxylamines) to form potent electrophiles is controlled by a specific gene.     

Nucleotide sequence and chromosomal location of Cab11 and Cab12, the genes for the fourth polypeptide of the photosystem I light-harvesting antenna (LHCI)

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned, cab11 and cab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome; cab11 on chromosome 3 and cab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chl a/b-binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma-exposed sequences preceding them. However, near the N-terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI-specific.     

Increase in β-1,4-Galactosyltransferase Activity During PC12 Cell Differentiation Induced by Forskolin and 2-Chloroadenosine

Galactosyltransferase (GALTase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chloroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine PC carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.     

Brain Micro vessel 12-Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.