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.     

Kinetics of formation of fibrin oligomers. II. Size distributions of ligated oligomers

Human fibrinogen was treated with thrombin in the presence of fibrinoligase (Factor XIIIa) and calcium ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) both before and after the clotting time (t_c) by solubilization with a solution of sodium dodecylsulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of γ-γ ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1 to 10, from the latter. In some experiments, thrombin was inhibited, after partial polymerization, by p-nitrophenyl-p′-guanidinobenzoate. From these, it was concluded that for thrombin concentration ?0.013 units/mL and fibrinoligase ?30 mg/L, oligomer assembly is rapid compared with peptide A release and ligation is rapid compared with assembly. Under these conditions, the theory of the first paper of this series describes rather well the time dependences of the degree of γ-γ ligation, the weight fractions of monomer and small oligomers, and the number- and weight-average degrees of polymerization after solubilization of the staggered overlapped assemblies, each of which splits to give two strands of end-to-end ligated oligomers. The theory assumes that the second A peptide is released by thrombin more rapidly than the first by a factor q, which, from the experimental data, is determined to be 16. The subsequent assembly into staggered overlapped oligomers follows the statistics of linear polycondesation taking into account the presence of both difunctional and monofunctional combining units. For higher thrombin or lower fibrinoligase concentrations, ligation fails to keep pace with oligomer assembly, and the size distributions after solubilization show a higher proportion of very small and a lower proportion of larger ligated oligomers, owing to separation of the staggered overlapped assemblies into smaller fragments.     

Application of the imidate procedure to α-d-galactosyltion

Using the imidate procedure, 2,3,4,6-tetra-O-benzyl-1-O-(N-methylacetimidoyl)-β-d-galactopyranose was condensed with various monosaccharides to provide, in good yield and with high stereoselectivity, α-linked disaccharides.     

Techniques for Preparing Seeds with Water-Impermeable Coats for Light and Electron Microscopy

An improved method for fixing and embedding seeds with impermeable coats for microscopic study has been developed. Entry portals are cut into seed coats to permit better penetration of fixative. This makes it possible to obtain semithin sections of whole seeds for light microscopy and thin sections of selected areas for electron microscopy. Seed tissues may thereby be studied relative to their position in the seed and to surrounding tissues. This permits studies of imbibition and developmental morphology of seeds in histological and cytological detail previously possible only with soft or dissected seeds.     

On the interaction of tritiated 2-ketobutyrate with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence suggesting enzyme-mediated,uncatalyzed tritium exchange

2-Keto-3-deoxygluconate-6P aldolase ofPseudomonas putida mediates exchange between hydrogen isotope at the methylene carbon of 2-ketobutyrate and water. This occurs with aK _m of 20 mM, 100 times the corresponding value for pyruvate, and a V_max approximating 1/710 that of KDPG cleavage. Ketobutyrate is competitive with both pyruvate and 2-keto-3-deoxygluconate-6P for the enzyme. In addition, there is no evidence for C-C synthesis between ketobutyrate andd-glyceraldehyde-3P. A comparison of relativeV/K values for hydrogen exchange shows pyruvate to be 17,600 times better as a substrate than ketobutyrate. The detritiation of [3-³H]ketobutyrate is stereochemically random. In addition, the reaction proceeds with ak _H/k _T isotope effect of 15.3, consistent with C-H bond turnover being rate-determining. The E-ketobutyrate complex is reductively trapped, inactivating the enzyme. Reductive inactivation kinetics of E-ketobutyrate compared to E-pyruvate suggests more of the complex may be partitioned to ketimine in the ketobutyrate case than in the pyruvate case. A mechanism is considered in which ketobutyrate is bound as a ketimine in an orientation such that the active site acid/basic group cannot mediate catalytic ketimine/eneamine interconversion. Thus, exchange would result from hydrogen ionization at C-3′ of the ketimine, a slow spontaneous step compared to overall complex turnover. This noncatalyzed deprotonation would explain dissymmetry in exchange, the poorV/K compared to pyruvate, and a large tritium isotope effect.     

The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.     

Reduced lipogenesis in cafeteria-fed rats exhibiting diet-induced thermogenesis

Fatty-acid synthesis has been measured in vivo with³H₂O in cafeteria-fed rats exhibiting diet-induced thermogenesis. Synthesis was decreased in brown adipose tissue, the liver, white adipose tissue, and the carcass of the cafeteria-fed animals compared to rats fed the normal stock diet. Whole-body synthesis was also decreased in the cafeteria-fed group. Diet-induced thermogenesis, in contrast to cold-induced non-shivering thermogenesis does not lead to increased fatty-acid synthesis and this is presumably due to the inhibitory effects on lipogenesis of the high dietary fat intake characteristic of cafeteria diets. The results also indicate that the energy cost of body fat deposition in cafeteria-fed rats is lower than in animals fed a low-fat/high-carbohydrate stock diet.