Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.     

Immunological study of a hemophiliac treated according to the Bonn protocol for 2 1/2 years

A major haemophiliac A, 27 years old, has been treated during 30 months, with high dosage of imported Factor VIII, in order to reduce the titer of a F VIII antibody. A good clinical result has been obtained. No sign of immunodeficiency has been observed. Normal values were obtained for T4/T8, ratio B2 microglobulin and no antibody was detected against the LAV virus isolated from cases of AIDS.     

Reduced hepatic alpha-tocopherol content after long-term administration of ethanol to rats

We have studied the effects of long-term administration of ethanol on the distribution and pharmacokinetics of alpha-tocopherol. In rats fed ethanol (35% of total energy) for 5-6 weeks concentration of alpha-tocopherol in whole liver was reduced by 25% as compared to the pair-fed controls (P less than 0.003). This reduction was significant in the parenchymal cells (28%, P less than 0.004), whereas no significant difference was observed for the nonparenchymal cells. Mitochondrial alpha-tocopherol content was reduced by 55% in the ethanol-treated rats as compared to the controls (P less than 0.002), whereas no significant difference was observed in microsomes, light mitochondria or cytosol. The serum levels of alpha-tocopherol showed no significant difference between the groups. When in vivo labeled chylomicron alpha-[3H]tocopherol was injected intravenously to anesthetized rats, we found a significant increase in serum half-life of alpha-tocopherol in the ethanol-treated group as compared to the controls (P less than 0.025). Hepatic alpha-[3H]tocopherol content was similar in the two groups 24 h after injection.