Role of the modulator protein in the interconversion of rabbit skeletal muscle protein phosphatase

The major active protein phosphatase present in a rabbit skeletal muscle extract is associated with the glycogen particle and migrates in sucrose density gradient centrifugation as a Mr = 70,000 protein and contains modulator activity. Addition of extra modulator protein causes a time- and concentration-dependent conversion of the enzyme to an inactive FA-ATP, Mg-dependent form. The intrinsic modulator in the active phosphatase is destroyed by limited proteolysis without an appreciable change in the phosphatase activity. The proteolyzed active enzyme has a lower molecular weight (Mr = 40,000) and it reassociates with the modulator producing a FA-ATP, Mg-dependent enzyme form (Mr = 60,000). The modulator protein is used stoichiometrically in the activation of the ATP, Mg-dependent phosphatase. This is in agreement with the presence of one unit of modulator activity per unit of native spontaneously active phosphatase.     

Optimal conditions for breaking medically important yeasts by an inexpensive and simple method

Conditions for breaking various medically important yeasts using glass beads, 30 ml Corex centrifuge tubes, and a Vortex mixer were determined. From 75–95% ofCandida hyphal cells and all species of yeasts exceptSporothrix schenckii were broken when 10 g of 0.45–0.50 mm glass beads, 50–300 mg of wet cells in 5 ml of buffer, and 90 s of vortexing were employed. Yeasts ofSporothrix schenckii broke more efficiently when 0.25–0.30 mm beads were used.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Andromonoecy and fruit set in three genera of the Proteaceae

It has been suggested by some authors that the low fruit to flower ratio in some Proteaceae is due to andromonoecy, while others, looking particularly at Banksia , have not been able to find evidence for male flowers in the inflorescences. Stirlingia latifolia, Xylomelum occidentals and X. angustifolium are clearly andromonoecous, while no evidence for this condition could be found in Brabejum stellatifoliutn. Production of fertile fruit is related to andromonoecy in Xylomelum and S. latifolia but not in Brabejum. It is unlikely that all-encompassing solutions will be found to what initially seem to be widespread traits in the family, especially in regard to pollination biology, as the genera in the family occupy widely different environments and have very diverse ecological ranges.     

The effect of hemoglobin ligands on the kinetics of human hemoglobin A1c formation

HbA1c is the most prevalent of the minor human hemoglobins. It is formed by the nonenzymatic addition of glucose to the alpha-amino group of the beta chain by an initial condensation reaction and a subsequent intermolecular Amadori rearrangement. We have developed a method of analysis which utilizes high performance liquid chromatography to follow the formation of HbA1c and greatly simplifies the determination of the kinetic parameters associated with this reaction. This has allowed us to study the effects of several Hb ligands, including the hydrogen ion, on the kinetics of this glycosylation reaction. Both the initial condensation reaction and the subsequent rearrangement are shown to exhibit acid catalysis, but the rate of the condensation step is limited by the extent of protonation of the alpha-amino group. The variation in kinetic parameters as a function of hydrogen ion concentration has allowed us to determine the probable reaction mechanism of HbA1c formation by comparison to previously reported model systems of Schiff base formation and Amadori rearrangement. The formation of pre-HbA1c from deoxy-Hb shows an increased forward rate when compared to oxy-Hb. The presence of physiologic concentrations of CO2 causes a proportional decrease in both k1 and k-1. 2,3-Diphosphoglycerate causes a significant increase in the keq of the formation reaction. The effects of CO and the substitution of L-glucose for D-glucose are not significant.     

Properties of purified colchicine-binding protein from a cultured carrot cell extract

Colchicine-binding protein (CBP) was purified from a cultured carrot cell extract by DEAE-Sephacel, phosphocellulose and Sephadex G200 column chromatographies. The purified CBP separated into three bands on SDS-polyacrylamide gel electrophoresis. One of them reacted with a monoclonal antibody against chick brain alpha-tubulin and the other two with that against beta-tubulin. Colchicine-binding activity of the purified protein was enhanced by tartrate and inhibited little by an excess of podophyllotoxin. It decayed following first order kinetics, but was more stable than the CBP in the crude extract. The binding constant of the purified CBP for colchicine was 0.57 microM-1 and the number of binding sites of colchicine per mg protein was about 2 nmol. This binding constant is about ten times lower than that of porcine brain tubulin under identical conditions.     

Characterization of beta-lactamase from Mycobacterium butyricum ATCC 19979

beta-lactamase has been purified to a homogeneous state from Mycobacterium butyricum ATCC 19979. The molecular weight (Mr = 29,000) and the isoelectric point (4,0) of the enzyme have been determined. The enzyme showed both penicillinase and cephalosporinase activity, but had relatively more of the former. With respect to substrate-profile the enzyme resembled the plasmid specified TEM-type beta-lactamases commonly encountered in Gram-negative bacteria. The enzyme was insensitive to p-chloromercuribenzoate, sodium chloride, or iodine inhibition.