Daniel H. Levin

The effects of 6-mercaptopurine on Lactobacillus casei

Abstract The inhibitory action of 6-mercaptopurine has been shown to be manifested by an interference with purine interconversion. The incorporation of adenine and of adenylic acid are affected in different ways by 6-mercaptopurine. These facts have been fitted to a schematic representation of a purine interconversion cycle compatible with the known metabolic behavior of L. casei.

[37] Double-stranded RNA-dependent eIF-2α protein kinase

Publisher Summary This chapter describes the purification and characterization of the latent form of the reticulocyte double-stranded RNA activated elF-2 α kinase (dsI). At all stages of purification of latent dsI, activation depends on dsRNA and ATP and is accompanied by a dsRNA-dependent phosphorylation of a polypeptide doublet (67K/68.5K) of dsI. Activated (and phosphorylated) dsI displays chromatographic properties on diethylaminoethyl (DEAE)-cellulose, phosphocellulose, and agarose poly(I) · poly(C) that are different from those of latent dsI although the molecular weight of dsI is unchanged by activation. The purification of a latent form of dsl facilitates further studies on the regulation of dsI activation and activity. There are several advantages in purifying and studying the latent form of dsI rather than the activated form: (1) latent dsI is significantly more stable and can be readily stored for prolonged periods, (2) examination of the molecular mechanism of activation and the function of dsRNA in this process is facilitated, and (3) the use of latent dsI facilitates studies on the regulation of dsI activation and activity by other cellular components, particularly protein phosphatases.

Characterization of a viral messenger ribonucleoprotein particle accumulated during inhibition of polypeptide chain initiation in reovirus-infected L cells

Abstract A prior study has shown that L cells deprived of essential amino acids in isotonic Tris-buffered KCl show a marked and rapid inhibition of protein synthesis which is due to a decrease in the rate of initiation of new peptide chains. The inhibition, which is manifested by a disaggregation of polysomes with apparent conservation of mRNA, is rapidly reversed when the cells are resuspended in complete growth medium. This phenomenon has been applied in studies on the mechanism of mRNA storage and reutilization in reovirus-infected L cells. When reovirus-infected L cells are incubated in KCl-Tris medium, the viral messengers accumulate as ribonucleoprotein particles which sediment at approximately 50 S in sucrose density gradients and have a density in CsCl of 1.40. These 50-S messenger-containing ribonucleoprotein particles do not appear to be complexed with ribosomal subunits. The particles contain all 10 species of viral mRNA and have no demonstrable viral proteins. The mRNA from these particles is readily and rapidly mobilized into functional polysomes when the infected cells are restored to normal growth medium. Moreover, the mRNA moiety of partially purified 50-S ribonucleoprotein particles can direct the incorporation of amino acids into protein in a cell-free system.

REGULATION OF EUKARYOTIC PROTEIN CHAIN INITIATION BY PROTEIN KINASES AND HEME

Publisher Summary This chapter describes the regulation of eukaryotic protein chain initiation by protein kinases and heme. Reticulocyte lysates provide a model system for studies on the regulation of protein synthesis owing to several unique properties. They include an efficient rate of initiation, the extensive synthesis of one major protein species, and the complete dependence of protein synthesis on exogenous hemin. In the presence of optimal concentrations of added hemin, protein synthesis proceeds at linear rates for prolonged periods. During the period of linear synthesis, the polyribosomes are maintained, and initiation factors are utilized catalytically. In efficient lysates, the rate of globin synthesis is comparable to the in vivo rate. In the absence of added hemin, protein synthesis proceeds at the control rate for several minutes, and then declines abruptly yielding characteristic biphasic kinetics of inhibition. The studies have established that the inhibition is mediated through a translational inhibitor which is rapidly activated in the absence of heme.

[32] 2-Keto-3-deoxy-8-phosphooctonic acid synthetase

Publisher Summary This chapter discusses the synthesis of 2-keto-3-deoxy-8-phosphooctonic acid synthetase (KPDO). The sugar KDPO belongs to a group of intermediates characterized as 2-keto-3-deoxy aldonic acids including KD-arabonic acid, KD-galactonic acid, KDP-gluconic acid, KDP-heptonie acid, and N-acetylneuraminic acid. The enzyme KDPO synthetase is purified from cell-free extracts of Pseudomonas aeruginosa pyocyaneus. Unfractionated extracts also contain KDPH synthetase, which catalyzes the condensation of D-erythrose 4-P and P-enolpyruvate. The two activities are separated by fractionation. The controlled oxidation of KD-aldonic acids by periodic acid yields β-formyl pyruvic acid, which arises from carbons 1 to 4 of the KD-aldonic acids because of a cleavage between carbons 4 and 5. The reaction of β-formyl pyruvic acid with 2-thiobarbituric acid results in the formation of a deep pink complex which displays a single peak in the visible spectrum at 549 mμ. Except where otherwise specified, all steps in the purification procedure are carried out at 0°–4°. The procedure described in the chapter is to remove KDPH synthetase and the epimerases that convert ribose-5-P and xylose-5-P to arabinose-5-P. The extent of purification achieved is 30-fold. Purification can also be achieved by fractionation on a diethylaminoethyl (DEAE)-cellulose column.