Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases

Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).     

Evidence for two forms of RNA-dependent DNA polymerase in Visna virus.

The visna viral RNA-dependent DNA polymerase has been resolved into two forms by affinity chromatography. Glycerine gradient centrifugation of the two forms showed that one form sedimented at 6.9 S corresponding to an apparent molecular weight of 135 000 and the other at 6.3 S corresponding to 118 000. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the two forms indicated that the 6.9 S enzyme is composed of 2 molecules of 68 000 mol. wt. chain and the 6.3 S is a single chain enzyme. The latter form has been identified as a glycoprotein. The 6.9 S form can be completely inactivated in 20 min at 45 degrees C, prefers poly(rC) over poly(rA) as template and has high efficiency in utilizing visna 70 S RNA as template. The 6.3 S form is stable at 45 degrees C, active with 70 S viral RNA as template, prefers poly(rA) over poly(rC), and requires higher concentration of Mn2+ (0.4 mM) for maximum activity than the 6.9 S form does (0.1 mM) with synthetic homopolymers as templates. However, both 6.9 S and 6.3 S forms prefer Mg2+ over Mn2+ regardless of the nature of the templates.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Streptozotocin treatment of streptozotocin-induced islet cell adenomas in rats.

Hypoglycemic rats bearing insulin-secreting islet-cell adenomas produced by the combined action of streptozotocin and nicotinamide were treated with streptozotocin. Antitumor response was demonstrated by elevation of blood glucose, reduction in plasma and tumor IRI, and histopathologic changes in the beta-cell neoplasm. The rodent tumor model may serve as a predictive system for selection and investigation of mechanisms of action of future antitumor agents to be used in the treatment of malignant insulinoma in man.     

Effect of propionate and pyruvate on citrulline synthesis and ATP content in rat liver mitochondria.

Propionate inhibits citrullinogenesis when succinate (plus rotenone) or glutamate are the oxidizable substrates used. Propionate decreases the intramitochondrial concentration of carbamylphosphate by decreasing the ATP content. When the energy supply for citrullinogenesis is provided by an influx of exogenous ATP, propionate is no longer an inhibitor. Pyruvate inhibits citrullinogenesis with glutamate but not with succinate (plus rotenone) as oxidizable substrates. Propionate and pyruvate deplete mitochondrial ATP but probably by different mechanisms.