The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" …

Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin …

A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.

Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.

The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.

N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the …

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild …

Mechanistic details on the regulation of striated muscle contraction still need to be determined, particularly the specific structural locations of the elements comprising the thick and thin filaments. Of special interest is the location of the regulatory component, troponin, on the actin filament and how its presence influences the behavior of myosin binding to the thin filament. In the present study: (1) Luminescence resonance energy transfer was used to monitor potential conformational changes in the reconstituted thin filament between the C-terminal region of troponin T and myosin subfragment 1; (2) Location of troponin in previously derived atomic models of the acto-myosin complex was mapped to visualize specific contacts; and (3) Shortened tropomyosin was engineered and protein binding and ATPase assays were performed to study the effect of myosin binding close to the troponin complex. Analysis of the results suggest the following: (1) Irrespective of calcium levels, the C-terminal region of troponin T is located close to myosin loop 3 and a few actin helices that may perturb strong acto-myosin interactions responsible for force production. (2) Atomic models indicate myosin subfragment 1 cannot attain the post- powerstroke state due to the full motion of the lever arm being sterically hindered by …

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint …

Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil …

The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial …

A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.

Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.

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.

A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully

O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.

The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.

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.

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.

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 …

A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.

Intracellular levels of NAD are of renewed interest in clinical and basic science research due to the new discovery of enzymes which utilize NAD as a substrate. Microtiter assays for the determination of NAD, NADP and protein were developed as modifications of previously published methods. The resulting assays are simple, cost effective and sensitive. An improved method of isolating lymphocytes was also developed. The resultant procedure requires one hour and removes greater than 99.9% of the platelets. Lymphocyte pools were stabilized with the addition of ADP-ribosyltransferase inhibitors and a modified extraction procedure. These studies have led to the development of a method for evaluation of NAD in human lymphocytes that is sensitive, selective and suitable for automation.

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.

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.