Partial Purification of Mucor pusillus Intracellular Proteases

The intracellular protease extracted from the freeze-dried mycelia obtained after the growth of Mucor pusillus at 30°C in corn steep liquor medium was chromatographed on DEAE-A50. Some characteristics of the protease fractions obtained after ion-exchange chromatography were determined and compared with those of the extracellular proteases reported previously. The mycelia were found to contain two acid proteases and an alkaline protease. The ratio of milk clotting to protease activity of one acid protease was greater than that of the other. The electrophoretic pattern of the alkaline protease fraction suggested that it was not a single species, but a mixture of proteolytic enzymes.     

Molecular Forms of Acetylcholinesterase: Regulation in a Testosterone-Sensitive Nerve-Muscle Axis

We measured the distribution of molecular forms of acetylcholinesterase (AChE) in muscles of a song bird, the zebra finch, and found a pattern similar to those reported in other vertebrates. As in other species, the most rapidly sedimenting form of the enzyme decreases to barely detectable levels following denervation. In the muscles of the syrinx, castration causes a large decrease in AChE activity, but has little or no effect on the relative abundance of AChE forms. This suggests that the number of AChE catalytic sites is changing without affecting the distribution of catalytic sites among the molecular forms. This is in marked contrast with the effect of denervation in the syrinx, which causes changes in the distribution of activity, as well as in total activity.     

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

Deductive analysis of a protein-synthesis mutant of Escherichia coli

A mutant of E. coli, T ^s68b, selected as unable to grow at 43 C, is unable to synthesize proteins at 43 C, though it can carry out this function at 30 C. This defect is shown to be in the protein-synthetic rather than the RNA-synthetic machinery by an analysis of the response of the strain to infection with the RNA bacteriophage f2. An analysis of the capacity for RNA synthesis and the polyribosome content of these cells at 44 C indicates that the defect resides in the elongation step of protein synthesis. No defects could be detected in vitro. The results are discussed in light of similar data on other mutants and in relation to the general approach of analyzing complex mutants selected with ill-defined phenotypes.This work was supported by Grant GM14368 from the National Institute of Health.     

THE EFFECT OF CULTURE CONDITIONS ON THE HYDROCARBON CONTENT OF CHLORELLA VULGARIS12

Cultures of Chlorella vulgaris were grown aulo-trophically under fluorescent light and heterotrophically on glucose and inorganic salts. Hydrocarbons were extracted and analyzed by gas-liquid chromatography, molecular sieve separations, and silicic acid-AgNO₃ chromatography. Chlorella vulgaris grown under both culture conditions contained a series of saturated n-paraffins ranging from 17 to 36 carbon atoms in length. This is in contrast to reports in the early literature which indicated that the hydrocarbon fraction of algae was composed of only 1 or 2 specific hydrocarbons. Only under heterotrophic conditions, however, did C. vulgaris produce 1-penta-cosene and 1-heptacosene as the primary components of the hydrocarbon mixture. Other Chlorella species were examined, but only C. vulgaris produced significant quantities of these compounds.