Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Ultracentrifugation in polyethylene tubing: An efficient method to concentrate protein solutions on the microliter scale

An ultracentrifugation procedure is described to concentrate protein solutions on the microliter scale. Protein solutions were centrifuged in U-shaped lengths of polyethylene tubing at 160 000 × g for 15 h and thereafter fractionated by cutting the tubing. The method can be performed in a conventional ultracentrifuge and needs no special equipment.     

Sympathetic nervous control of blood flow in the gills of the Atlantic cod,Gadus morhua

Summary The effects of sympathetic nerve stimulation, adrenaline and isoprenaline on the inflow pressure and efferent arterial and venous flow rates were studied in a cod gill preparation perfused at constant flow rate.The dominant effect of adrenaline was a reduced inflow pressure, accompanied by an increase in arterial flow and a decrease in venous flow. Isoprenaline also decreased the inflow pressure, but the changes in both outflow rates were small or absent.Sympathetic nerve stimulation gave arterial and venous flow changes comparable to the adrenaline effects, but the inflow pressure increased during nerve stimulation. Propranolol has little effect on the nerve responses, but phentolamine abolished or reversed the increase in inflow pressure, and also decreased or abolished the changes in outflow rates.The possible sites of action of the sympathetic fibres, and the distribution of adrenoceptors in the effector tissue is discussed. It is concluded that the main effect of sympathetic nerve stimulation is -adrenoceptor mediated, involving constriction of the arterio-venous pathway. The-adrenoceptor mediated control of total branchial vascular resistance may largely depend on circulating catecholamines.     

The Sequence of Events in the Development of Bilateral Renal Cortical Necrosis Accompanying the Generalized Shwartzman Reaction

The generalized Shwartzman reaction, or Shwartzman-like conditions, were induced in a variety of experimental mammalian species by systemic injections of disintegrated cells of Gram negative bacteria, live Salmonella cholerae-suis or Liquoid. A comparative study of the renal lesions showed that the initial step in the development of bilateral cortical necrosis is stagnation and disintegration of red cells in glomerular capillaries. The glomerular “microthrombi” consist mainly of erythrocytic debris, which frequently has staining properties akin to those of fibrin; even wide-spread glomerular “thrombosis” is not accompanied by obvious destruction of renal parenchyma. A second step is necrotic mural lesions in afferent arteries, with ensuing thrombosis. These vascular lesions lead to the formation of individual infarcts which fuse to form total bilateral cortical necrosis in fulminant cases of the generalized Shwartzman reaction.     

Influence of Diet on Experimental Swine Dysentery. 1. Effects of A Vitamin E and Selenium Deficient Diet Supplemented with 6.8 % Cod Liver Oil

Sixteen growing pigs were fed a vitamin E and selenium deficient diet; half of the animals (Group 2) were given a daily supply of vitamin E and selenium. After having been fed these diets for 53 days, the pigs were infected orally with minced colonic material from cases with typical swine dysentery. This exposure resulted in outbreaks of swine dysentery in both groups. The incubation times were, however, distinctly shorter and the clinical symptoms much more pronounced in Group 1 than in Group 2. The patho^morphological lesions in the colon also differed between the 2 groups. In the pigs of Group 1 evident pseudomembraneous lesions were observed in the spiral colon. In Group 2, the colonic alterations consisted predominantly of a catarrhal enteritis; pseudomembranes occurred in a minor part of colon in only 4 pigs. Both the clinical and the chemical observations and the pathological findings indicated a much better vitamin E and selenium balance in the pigs of Group 2. It is concluded that the treatment with vitamin E and selenium in Group 2 greatly increased resistance to swine dysentery.     

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH₂CH(X)COOCH₃ > RCH₂CH(X)CONH₂ > RCH₂CH(X)COOH > RCH₂CH₂X > RCH₂CH(X)CH₂OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS^?) is the reactive species, but only minor differences emerged between different free thiolates.     

Electron Microscopic Changes in Colon in Experimental Swine Dysentery

Colonic lesions in experimental swine dysentery were studied electron microscopically. Changes indicative of stasis were commonly observed in the microcirculatory vessels of lamina propria. Early lesions in epithelial cells included sparse, short and irregular microvilli, swollen and degenerated mitochondria, and swelling and vesiculation of endoplasmatic reticulum. Numerous large spirochaetes were observed in these locations: a) in the crypts, b) free (i.e. not membrane bound) in cytoplasm of damaged epithelial cells, and c) in cavities, around vessels of lamina propria. It is suggested that stasis, and resultant disturbances in microcirculation in early developmental stages of swine dysentery, may play a pathogenetic role in the development of the necrotic colonic lesions. Finally, it is discussed whether a mechanism related to Sanarelli-Shwartzman reaction is implicated in the development of colonic lesions in swine dysentery.