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.

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.

A description of the nature of the transition state structure for phosphoryl transfer in the cAPK reaction requires a measurement of the primary 180 isotope effect at the serine hydroxyl acceptor. Since it is difficult to obtain primary 180 isotope effect directly, the 15N/1 4N ratio of the a-amine of the C-terminal glycine in the peptide Leu Arg-Lys-Ala-Ser-Leu-Gly (when serine is phosphorylated) was used to represent on the phosphorylation at serine. 15N Glycine, ' 4N-Glycine and 180 serine were synthesized and used to synthesize two peptides, one containing 1 80-serine/' 5 N glycine and second 1 60-serine/1 4N-glycine. Methods were developed for hydrolyzing the peptides and quantitatively isolating glycine. Partitioning results suggest that catalytic rate was slow compare to substrate dissociation. The 180 primary isotope effect will be determined in the near future using the method developed herein.

Two plaque-pure phage lambda clones designated as λhtX-l and λhtX-2 that hybridized to unfractionated bovine liver tRNA were isolated from a human X chromosome-specific library. The λDNAs were characterized by restriction mapping and Southern blot hybridization techniques. The human DNA segment in λhtX-l contains five or more presumptive tRNA genes and at least one Alu family member. The 19-kilobase human DNA insert in λhtX-2 contains two or more presumptive tRNA genes and at least three Alu family members. Another human genomic clone designated λhVKV7 hybridized to mammalian valine tRNA IAC. The clone was characterized by physical mapping and Southern blot hybridization techniques. The 18.5-kilobase human DNA fragment in λhVKV7 contains a cluster of three tRNA genes and at least nine Alu family members.

Glyoxylase II (Glo II, E.C. 3.1.2.6) catalyzes the hydrolysis of S-D-Lactoylglutathione (SLG) to D-Lactate and glutathione. This is the rate limiting step in the conversion of methylglyoxal to D-Lactate. The purpose of the present study was to determine whether or not a relationship exists between some naturally occuring metabolites and in vivo modulation of Glo II. We have observed a non-competitive inhibition (~ 45%) of Glo II in crude preparation of rat liver by GTP (0.3 mM). A factor (apparently protein),devoid of Glo II,when reconstituted with the purified Glo II, enhanced Glo II activity. This coordinate activation and inhibition of Glo II suggest a mechanism whereby SLG levels can be modulated in vivo.

Tyrosine tRNA was isolated from bovine liver and its nucleotide sequence was determined using in vitro 32p_ labeling techniques. Several important structural features of the tRNA are: the presence of gal-Q in the first position of the anticodon, acp3U at position 20, and a pair of adjacent N,N-dimethylguanosines (residues 26 and 27). A human DNA fragment harbored in a lambda phage clone was isolated, and restriction enzyme analysis revealed the presence of three tRNA genes in a 6.0-kb BamHI subfragment. Portions of the 6.0-kb DNA fragment containing the tRNA genes were sequenced by the method of Maxam and Gilbert and analyzed for transcriptional activity in vitro using homologous cytoplasmic extracts. A threonine tRNAUGU gene exhibited high transcriptional activity dependent on its 5'- flanking sequence. The enhanced transcription is not completely inhibited by alpha-amanitin. The value of studying tRNA structure in concert with the cognate tRNA. genes is discussed.

Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.

DL-2-Amino-4-phosphonobutyric acid, an isostere of phosphoserine, was incorporated into the heptapeptide sequence, Leu-Arg-Arg-Ala-(DL-2-amino-4-phosphonobutyric acid)-Leu-Gly, for kinetic mechanistic studies of the cAMP-dependent protein kinase. To block the phosphono hydroxyl groups, methyl, ethyl and 4nitrobenzyl esters were studied as possible protecting groups. The phosphono diethyl ester of the N-Fmoc-protected amino acid was utilized in the synthesis of the heptapeptide. Two configurational forms of the protected peptide were obtained and were separated by C18-reverse phase HPLC. Characterization of the two isomeric forms was accomplished by 3 1P NMR, 1H NMR, 13C% NMR and amino acid analysis. The protecting groups of the isomeric phsophonopeptides were removed by HBr/AcOH and purified by cation exchange HPLC. Both phosphonopeptides were found to be inhibitors of the cAMP-dependent protein kinase, having Ki values of 0.6 mM (peptide A) and 1.9 mM (peptide B).

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.

Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.

A method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product.

Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.

Glyoxalase 11, the second enzyme of the glyoxalase system, hydrolyzes S-D-lactoylglutathione (SLG) to regenerate glutathione (GSH) and liberate free D-lactate. It was found that GTP binds with Gil from rat liver and inhibits Gil activity. Preincubation experiments showed that the binding is relatively tight, since more than 15 minutes are required to release GTP from the complex following dilution. Inhibition kinetics studies indicate that GTP is a "partially competitive inhibitor"; Thus, it would appear that the binding sites for substrate (SLG) and inhibitor (GTP) are different, but spatially close. Glyoxalase 11 binds to a GTP affinity medium, and with polyacrylamide gel electrophoresis, Gil has a higher relative mobility when GTP is present (ATP has no effect). The functional consequences of GTP binding with a specific site on Gil are still unclear. It is speculated that Gil may interact with tubulin by serving as a dissociable GTP carrier, delivering GTP to the tubulinGTP binding site, and thus facilitating tubulin polymerization.

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.

Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism …

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence …

Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of …

The mechanism by which nicotinamide adenine dinucleotide (NAD) stimulates the activity of adenylate cyclase (AC) in canine plasma membrane has been studied. Using [3 2P]-NAD, the activation by NAD was correlated with the radiolabeling of the stimulatory guanosine triphosphate (GTP) binding protein Gsa. Further characterization demonstrated that the modification occurred only in the presence of G-protein activators and that arginine residue(s) were modified by ADP-ribose by the action of a mono-ADP-ribosyltransferase. Inhibitors of the transferase blocked both the modification of Gsa and the activation of AC. Collectively, these studies suggest that ADP-ribosylation of Gsa by an endogenous mono-ADP-ribosyltransferase may regulate cardiac AC.

A novel molecular mechanism of autophosphorylation-dependent activation of the ser/thr S6/H4 kinase isolated from human placenta is described. Phosphopeptide mapping of the enzyme was used to determine the rate and extent of site-specific autophosphorylation. These data were correlated to phosphotransferase activity of the protein kinase. The results indicated that a sequential phosphorylation of two sites in the catalytic domain is required for maximum activation. Kinetic analysis determined that site 1 is modified by an intramolecular phosphorylation, and site 2 is modified by an intermolecular phosphorylation. On the basis of these data a model is proposed in which autophosphorylation of the pseudosubstrate domain and on a serine residue in subdomain VIII are both required for maximum activation of the S6/H4 kinase.

Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was …

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.

Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.

The kinetic mechanism of the cAMP-dependent protein kinase has been determined to be random in the direction of MgADP phosphorylation by using initial velocity studies in the absence and presence of the product, phospho-Serpeptide (Leu-Arg-Arg-Ala-Ser[P]-Leu-Gly) , and dead-end inhibitors. In contrast to the kinetic parameters obtained in the direction of Serpeptide phosphorylation, the only kinetic parameters affected by Mg^2+ are the dissociation constants for E:phospho-Serpeptide and E:MgADP, which are decreased by about 4-fold. The dead-end analog MgAMPCP binds with an affinity equal to that of MgADP in contrast to MgAMPPCP, which binds weaker than MgATP. The ratio of the maximum velocities in the forward and reverse reactions is about 200, and the Haldane relationship gives a K-eq of (7.2 ± 2) x 10^2. The latter can be compared to the K-eq obtained by direct measurement of reactant concentrations (2.2 ± 0.4) x 10^3 and 31-P NMR (1 ± 0.5) x 10^3. Data for the pH dependence of kinetic parameters and inhibitor dissociation constants for the cAMP dependent protein kinase are consistent with a mechanism in which reactants selectively bind to an enzyme with the catalytic base unprotonated and an enzyme group required protonated for Ser-peptide binding. Preferentially MgATP binds fully …

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of …