Interaction of chlorambucil with tRNA.

Preincubation of purified mixed tRNAs from Escherichia coli K12-MO with 2.94 mM chlorambucil (CAB) for 2 h at 37 degrees C results in the inhibition of the capacity of mixed tRNAs to accept alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine by 100, 71, 100, 100, 100, 95, 32, 88, 36, 26, 96, 78, 44, 31, 34, 98, 38, and 17% respectively. Preincubation of tRNA with 0.75 mM and 0.29 mM CAB inhibited aminoacylation by aspartic acid to the extent of 69 and 17% respectively. CAB has no apparent effect upon the capacity of ATP to function in the formation of aminoacylated tRNALeu.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Complete replication of DNA in polytene nuclei of salivary glands ofDrosophila melanogaster

Summary Combined cytophotometric and autoradiographic experiments are performed on individual polytene salivary gland nuclei of X/X-female and X/Y-male larvae ofDrosophila melanogaster, DNA measurements of unlabeled nuclei reveal complete douplings of all 4C DNA quantity during polytenization. These new data do not agree with the hypothesis of heterochromatic underreplication.     

The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis.

Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.