Q-Beta phage RNAs with inactivating insertion (8 base) or deletion (17 base) mutations within their replicase genes were transfected into Escherichia coli spheroplasts containing QB replicase provided in trans by a resident plasmid. Replicase-defective (Rep~) Q3 phage produced by these spheroplasts were unable to form plaques on cells lacking this plasmid. When individual Rep~ phage were isolated and grown to high titer in cells containing plasmid derived Q3 replicase, revertant Q3 phage (Rep'), with the original mutation (insertion or deletion) repaired, were obtained at a frequency of ca. 1 x 108. RNA recombination via a "template switching" mechanism involving Q3 replicase, the mutant phage genome, and the plasmid-derived replicase mRNA was shown to be the primary means by which these mutant phages reverted to wild type.

Modification of proteins by ADP-ribose has been shown to be a versatile modification with respect to the amino acid side chain. The results described here will allow the study of the biological importance of ADP-ribose glycation and also allow differentiation on crude extracts between enzymatic modifications from protein ADP-ribose glycation that can occur due to the presence of NAD glycohydrolases.

Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.

To define the overall kinetic and chemical mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the mechanism in the direction of MgADP phosphorylation was determined, using studies of initial velocity in the absence and presence of dead-end inhibitors. The kinetic mechanism was determined as a function of uncomplexed Mg^2+ (Mg_f) at pH 7.2 and as a function of pH at low (0.5 mM) Mg_f. At pH 7.2 data are consistent with a random kinetic mechanism in the direction of MgADP phosphorylation with both pathways allowed: the pathway in which MgADP binds to enzyme prior to phosphorylated peptide (PSP) and that in which PSP binds before MgADP. One or the other pathway predominates, depending on Mg_f concentration. At 0.5 mM Mg_f, the mechanism is steady-state ordered with the pathway where PSP binds first preferred; at 10 mM Mg_f, the mechanism is equilibrium ordered, and the pathway in which MgADP binds first preferred. This change in mechanism to equilibrium ordered is due to an increase in affinity of enzyme for MgADP and a decrease in affinity for PSP. There is also a pH-dependent change in mechanism at 0.5 mM Mg_f. At pH 6 the mechanism is equilibrium ordered with the pathway …

The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.

Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.

This study examined how the monoblast-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristrate 13-acetate (PMA) to undergo differentiation. In order to study the mechanism of action of CSF-1, a CSF-1 receptor gene (c-fms) was transfected into U937 cells. Exogenous CSF-1 treatment induced an autocrine response in this CSF-1 was determined and all events were shown to be time dependent. CSF-1 stimulation also enhanced proto-oncogene c-jun and c-myc gene expression. Complementary DNA coding for Jun or Fos was introduced into U937 cells by transfection. The transfection did not generate a high level of CSF-1 gene expression which suggests that Fos and Jun alone are insufficient to induce CSF-1 synthesis.