Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher …

This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in …

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

Glucose phosphate isomerase (GPI) was purified from human placenta utilizing cross-linked spherical particle phosphocellulose. In three steps, GPI could be purified approximately 5500 fold with greater than 50% recovery. The purified enzyme exhibited four bands upon non-denaturing PAGE and isoelectric focusing (IEF) when stained with GPI specific activity stain. The four isozymes were isolated by preparative IEF. The isoelectric points of the isozymes were determined. Sodium dodecyl sulfate (SDS) gel electrophoresis showed two types of subunits with different molecular weights. Structural analyses showed both types of subunits had blocked amino termini. Other properties of the isozymes and subunits, including immunological reactivity, pH stability, peptide mapping and amino acid composition, were also established.

Lecithin:cholesterol acyltransferase (LCAT) was isolated from hog plasma and basic physicochemical properties and functionally important regions were investigated. Approximately one milligram of the enzyme was purified to apparent homogeneity with approximately a 20,000-fold increase in specific activity. In the plasma, hog LCAT was found to associate with high-density lipoproteins (HDL) probably through hydrophobic interactions with apolipoprotein A-I. HDL was the preferred lipoprotein substrate of the enzyme as its macromolecular substrate. The enzyme was found to contain 4 free sulfhydryl groups; at least one of these appeared to be essential for catalytic activity. The enzyme had a tendency to aggregate at high concentrations. More than half of the tryptophan and none of the tyrosine residues of the enzyme were shown to be exposed to the aqueous environment based on fluorescence and absorbance studies, respectively.

This study demonstrates the presence of protein kinase C activity in both cytosolic and membrane fractions of bovine lens epithelial cells in culture. Protein kinase C activity is similar in normal and hyperglycemic cells. Furthermore, the ability of the enzyme to translocate from the cytosol to the membrane following phorbol ester treatment is unimpeded by hyperglycemic conditions. Moreover, protein kinase C activation had no effect on myoinositol uptake either in normal cells or in cells exposed to hyperglycemic conditions.

Methodology was developed which allowed the rapid and routine quantitation of subpicomole quantities of diadenosine-5ʹ,5ʹʹʹ-P¹,P⁴-tetraphosphate (Ap₄A) in cultured mammalian cells. This methodology includes the rapid extraction of cellular nucleotides in cold alkali, resolution of Ap₄A from the bulk of cellular materials on a highly specific boronate affinity resin, and quantitation of the dinucleotide in a coupled bioluminescence assay utilizing venom phosphodiesterase and firefly luciferase. The sensitivity and selectivity of this assay is demonstrated and contrasted with previously developed techniques. This assay was used to examine the role of Ap₄A in DNA replication and the cellular stress response.

ADP-ribose polymer metabolism has been studied in human cells derived from a patient with Glutamyl Ribose Phosphate Storage Disease (GRPSD) and in mouse C3H1OT1/2 cells following oxidative stress induced by hydrogen peroxide (H202 ). It has been postulated that GRPSD resulted from an abnormality in ADP-ribose polymer metabolism. This study has shown that these cells exhibit reduced poly(ADP ribose) polymerase activity which is proposed to result from modification of the enzyme with ribose phosphate groups. The modification in the polymerase is proposed to be secondary to a defect in either ADP-ribosyl proteinlyase or an overproduction of a cellular phosphodiesterase. The metabolism of ADP-ribose polymers was rapidly altered by H202 and there were independent effects on adenine nucleotide pools. The results suggest that ADP-ribose polymer metabolism is involved in cellular defenses to oxidative stress.

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.