Substrate Kinetics Research Articles

The Role of DNA “Bendability” in the Indirect Read-Out Mechanism of Protein-DNA Interactions

Integration host factor (IHF) from E. coli is a DNA-bending protein that recognizes and binds to its specific sites primarily by the indirect read-out mechanism, in which sequence-dependent DNA dynamics and flexibility play an important role. The crystal structure of IHF bound to a 35-bp long cognate site H' indicates that the DNA is kinked at two sites separated by ∼9 bp, resulting in a “U-turn” bend of the DNA. We use laser temperature-jump to perturb the IHF-DNA complex, and time-resolved FRET on end-labeled DNA substrates to monitor the bending/unbending dynamics. In our previous studies, we suggested that spontaneous DNA bending from transient disruption of base-pairing and/or stacking interactions at the site of the kinks may be the rate-limiting step in the transition from the nonspecific to the specific complex. Here, we investigate DNA bending kinetics for substrates with mismatched pairs introduced at the site of the kinks. These internal “loops” are expected to decrease the energetic cost of bending the DNA, which is reflected in the >10-fold increase in the binding affinity. Kinetics measurements on IHF bound to such DNA reveal deviations from single exponential relaxation, indicating two distinct phases. Of particular interest is the observation that the rapid phase has a rate that is 10-20 times faster than the bending rate observed in the IHF-H' complex. Thus, reducing the energetic cost of bending/kinking DNA speeds up the bending rate by nearly the same factor as the increase in binding affinity, indicating that the free energy of the transition state is lowered by the same amount as the free energy of the complex. These results support our earlier conclusion, that spontaneous bending of DNA is the first step in the recognition mechanism.

Evidence of parietal amine oxidase activity in Solanum torvum Sw. stem calli after Ralstonia solanacearum inoculation

Calli induced from Solanum torvum stem explants were inoculated with Ralstonia solanacearum under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H(2)O(2) production was detected in semi-purified cell walls of calli infiltrated by the bacteria. Due to its preferential affinity for monoamines, this enzyme is supposed to have monoamine oxidase-like (MAO-like) activity. Moreover, the presence of hydroxyl radicals in the aromatic cycle alters the oxidative deamination kinetics of potential substrates. Indeed, the oxidation of dopamine (+2, OH) was shown to be faster than that of tyramine (+1, OH), which in turn was faster than that of phenylethylamine (0, OH). The MAO-like catalytic activity was significantly inhibited by some reducing agents such as sodium bisulphite and cysteine, and also by tryptamine under anaerobiosis. This latter result suggested that the prosthetic group of the MAO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure. Finally, the sigmoid kinetics of the MAO-like enzyme in semi-purified cell walls did not correspond to that expected for a purified MAO, suggesting that the kinetics were affected by some factors present in cell walls.

Effect of treatment with cadmium on kinetic properties of Na +, K +-ATPase and glucose-6-phosphatase activity in rat liver microsomes : A correlative study on influence of lipid/phospholipid make-up

Studies on Cd hepatotoxicity have focused mainly on induction of cytochrome P 450 system and related enzymes. In the present study young adult male rats given a single intra-peritoneal injection of Cd (0.84 mg Cd/kg body weight) and effects on kinetic parameters rat liver microsomal Na +, K +-ATPase and G6Pase were evaluated at the end of 1 month and 1 week. The substrate and temperature kinetics parameters were examined and attempts were made to seek correlation with changes in lipid/phospholipid profiles. The Na +, K + ATPase activity decreased only in 1 week Cd-treated group but recovered at the end of 1 month. The activity resolved in two distinct kinetic components in control as well as the experimental groups. In 1 week Cd-treated group the K m value of both the components was unchanged, whereas V max value decreased. In 1-month Cd-treated group V max value only of component I increased. The catalytic efficiency of both the components was not affected in the experimental groups. In 1-week Cd-treated group the energy of activations at high-temperature range ( E H) and low-temperature range ( E L) decreased, whereas for 1-month Cd-treated group the energies of activations did not change. The G6Pase activity measured at 37 °C was high only in 1-month Cd-treated group. The activity resolved in two kinetically distinguishable components in control as well as in the experimental groups. K m value of component I decreased in both the Cd-treated groups. In 1-month Cd-treated group the V max value of component II increased. The catalytic efficiency of G6Pase was unchanged despite changes in K m and V max. In 1-week Cd-treated group the E H and E L decreased, whereas only E L showed decrease in 1-month Cd-treated group. Cholesterol (CHL) content increased in both the Cd-treated groups. Content of lysophospholipid (Lyso), spinghomyelin (SPM) and phosphatidic acid (PA) increased, whereas phosphatidylcholine (PC) and phosphatidylserine (PS) decreased in 1-week Cd-treated group. In 1-month Cd group the Lyso, SPM, and PC increased while PC, phosphatidylethanolamine (PE) and PA decreased. In conclusion, Cd has short-term effects on microsomal Na +, K +-ATPase which are reversed by the end of 1 month and that G6Pase does not seem to be a target of Cd insult.

Modelling the industrial production of vinegar in aerated-stirred fermentors in terms of process variables

The production of vinegar in an aerated-stirred reactor was modelled using a hybrid approach. The growth rate was modelled according to the total biomass specific growth rate, μ g ν , which represents the growth of an active biophase and the fraction of cells which are growing. A quadratic model based on the process variables, i.e. hydrostatic pressure, ethanol concentration, aeration, agitation and temperature, was developed for μ g ν and the substrate and product kinetics were related mechanistically to the growth rate, including the kinetics of acetoin and ethyl acetate production. This model was coupled with the models for oxygen transfer and gas–liquid transfer, reported in a previous study, and the prediction capacity of the system of equations was validated by simulating semi-continuous and continuous real processes developed in a pilot fermentor.

Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.

Thyroidal regulation of substrate kinetics properties of cytochrome oxidase in rat liver mitochondria

Effects of thyroidectomy (T(x)) and subsequent treatment with 3,5,3'-triiodothyronine (T(3)), and combined treatment (T(R)) with T(3) + thyroxine (T(4)) on substrate kinetics properties of cytochrome oxidase of rat liver mitochondria were examined. T(x) resulted in lowering of cytochromes content with decrease in the enzyme activity, and K(m) and V(max). T(3) and T(R) regimens restored the cytochromes contents and the V(max) values to normal. In control, T(3) and T(R) groups the enzyme activity resolved in two kinetic components; in T(x) group three kinetic components were evident. The K(m) values for all components decreased significantly in the experimental groups with concomitant increase in catalytic efficiency, K(cat)/K(m). Significant alterations in the contents of total phospholipid and of cholesterol were noted while the changes in the phospholipids composition were only of restricted nature. Regression analysis revealed that total phospholipid, cholesterol and phosphatidylcholine, phosphatidylethanolamine play significant role in fine tuning the enzyme activity.

Transport of amino acids and GABA analogues via the human proton-coupled amino acid transporter, hPAT1: Characterization of conditions for affinity and transport experiments in Caco-2 cells

The objective of this study was to investigate transepithelial amino acid transport as a function of Caco-2 cell culture time. Furthermore, the objective was to investigate apical uptake characteristics of hPAT1-mediated transport under various experimental conditions. Apical amino acid uptake and transport studies were conducted in Caco-2 monolayers cultured for 4–28 days. Transepithelial transport of the prototypic hPAT1 (SLC36A1) substrates l-proline and glycine were maximal after 21–28 days in culture. Based on proton-dependency and substrate kinetics the major apical uptake and transport of Gly and Pro in Caco-2 cell monolayers is hPAT1-mediated. The apical uptake of Pro is decreased at apical hyperosmolarity conditions. Furthermore we identified the two GABA-analogues, muscimol and THPO as novel hPAT1 substrates. THPO had an affinity for hPAT1 of 11.3 mM, whereas muscimol had one of the highest affinities for hPAT1 (1.7 mM) reported. Our findings illustrate the suitability of the Caco-2 model for studying hPAT1-mediated transport. Furthermore, the affinity of THPO and muscimol underlines the possible importance of hPAT1 as a transporter for heterocyclic compounds consisting of a 3-isoxazolol moiety, which has been shown to function as a carboxylic acid bioisostere for substrates of the GABA receptor and transport systems.

Angiotensin‐converting enzyme‐like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.

Aging, Aluminium and Basal Forebrain Lesions Modify Substrate Kinetics of Erythrocyte Membrane Na,K-ATPase in the Rat

Several studies suggested that the activity of erythrocyte Na,K-ATPase declines with aging. Here, it is postulated that alterations in the substrate kinetics of the erythrocyte membrane Na,K-ATPase could be more aggravated in conditions of brain cholinergic dysfunction seen in Alzheimer's disease than in normal aging. To test this hypothesis, we compared the Na,K-ATPase activity (Vmax/Km parameters) in aged rats with those in young rats with brain cholinergic dysfunction induced by electrolytic-, kainic acid-lesioned nucleus basalis magnocellularis (NBM) or by intracerebroventricular AlCl_{3} administration. In the above mentioned groups, Vmax values were significantly lower in comparison to the control animals. Furthermore, Km values were significantly higher in animals with electrolytic-induced NBM lesions, AlCl_{3} treated rats and aged animals. However, Km was significantly lower in kainic acid-induced NBM lesions compared to the control group. The Na,K-ATPase catalytic efficiency, estimated by the ratio Vm/Km, decreased as followed: young animals > aged animals > kainic acid lesion > electrolityc lesion > AlCl_{3}. Our data suggest that neurodegenerative processes similar to those seen in Alzheimer's disease affect the sodium/potassium pump functionality which might be detected in peripheral blood erythrocyte membranes.

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.

Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity

Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.

Alternative Catalytic Anions Differentially Modulate Human α-Amylase Activity and Specificity,

A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic alpha-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic alpha-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic alpha-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.

Influence of mutations associated with Gilbert and Crigler–Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates

UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances. The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

The Dinuclear Zn(II) Complex Catalyzed Cyclization of a Series of 2-Hydroxypropyl Aryl Phosphate RNA Models: Progressive Change in Mechanism from Rate-Limiting P−O Bond Cleavage to Substrate Binding

A methoxide-bridged dinuclear Zn(II) complex of 1,3-[N,N'-bis(1,5,9-triazacyclododecane)]propane (1-Zn(II)2:(-OCH3)) was prepared, and its catalysis of the cyclization of a series of 2-hydroxypropyl aryl phosphates (4a-g) was investigated in methanol at pH 9.8, T = 25degreesC by stopped-flow spectrophotometry. An X-ray diffraction structure of the hydroxide analogue of 1-Zn(II)2:(-OCH3), namely 1-Zn(II)2:(-OH), reveals that each of the Zn(II) ions is coordinated by the three N's of the triazacyclododecane units and a bridging hydroxide. The cyclizations of substrates 4a-g reveal a progressive change in the observed kinetics from Michaelis-Menten saturation kinetics for the poorer substrates (4-OCH3 (4g); 4-H (4f); 3-OCH3 (4e); 4-Cl (4d); 3-NO2, (4c)) to second-order kinetics (linear in 1-Zn(II)2:(-OCH3)) for the better substrates (4-NO2,3-CH3 (4b); 4-NO2, (4a)). The data are analyzed in terms of a multistep process whereby a first formed complex rearranges to a reactive complex with a doubly activated phosphate coordinated to both metal ions. The kinetic behavior of the series is analyzed in terms of change in rate-limiting step for the catalyzed reaction whereby the rate-limiting step for the poorer substrates (4g-c) is the chemical step of cyclization of the substrate, while for the better substrates (4b,a) the rate-limiting step is binding. The catalysis of the cyclization of these substrates is extremely efficient. The kcat/KM values for the catalyzed reactions range from 2.75 x 10(5) to 2.3 x 10(4) M-1 s-1, providing an acceleration of 1 x 10(8) to 4 x 10(9) relative to the methoxide reaction (k2OCH3, which ranges from 2.6 x 10(-3) to 5.9 x 10(-6) M-1 s-1 for 4a-g). At a pH of 9.8 where the catalyst is maximally active, the acceleration for the substrates ranges from (1 - 4) x 10(12) relative to the background reaction at the same pH. Detailed energetics calculations show that the transition state for the catalyzed reaction comprising 1-Zn(II)2, methoxide, and 4 is stabilized by about -21 to -23 kcal/mol relative to the transition state for the methoxide reaction. The pronounced catalytic activity is attributed to a synergism between a positively charged catalyst that has high affinity for the substrate and for the transition state for cyclization, and a medium effect involving a reduced polarity/dielectric constant that complements a reaction where an oppositely charged reactant and catalyst experience charge dispersal in the transition state.

Deoxycytidine Kinase Genotype Influences Leukemia Cell Concentration of Cytarabine 5′-Triphosphate in Pediatric AML Patients.

Cytarabine (Ara-C) is a prodrug requiring conversion to its 5′-triphosphate (ara-CTP) form for antileukemic effect. Alteration of deoxycytidine kinase (DCK), a rate-limiting enzyme in this conversion, is associated with ara-C resistance. We sought to identify and determine the functional and clinical significance of DCK genetic variants. Sixty-four genetic polymorphisms were identified, including 3 coding changes (Ile24Val, Ala119Gly, and Pro122Ser amino acid substitutions), in subjects of European or African ancestry. The activity of recombinant DCK-24Val, DCK-119Gly, and DCK-122Ser was 85±5%, 66±3%, and 43±4%, respectively, that of wild-type (WT) DCK. These 3 DCK mutants also demonstrated altered substrate kinetics. Lymphoblast cell lines derived from subjects heterozygous for these coding changes had DCK activity significantly lower than that of homozygous-WT lymphoblasts (176.7±15 vs. 340.8±19 pmol CdA-MP/min/mg protein; p=0.0004). Moreover, for a 3′UTR 35708T>C SNP, the C allele was significantly associated with lower DCK mRNA expression in lymphoblast cell lines (p=0.02). To determine the clinical implications of DCK SNPs, we screened pediatric AML patients treated on the St Jude AML 97 protocol (n=55) for the 3 coding variants and the 3UTR SNP. Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hours after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher in patients given a short daily infusion than in those given a continuous infusion (0.56±0.5 vs. 0.33±0.31 nmol ara-CTP/2x107 leukemia cells; p = 0.014). The inter-patient variability of blast ara-CTP concentration was 40-fold (0.06–2.43 nmol/2x107 cells) when ara-C was administered as short infusion and 101-fold (0.012–1.22 nmol/2x107 cells) when given as a continuous infusion. Karyotype, sex, age, and ethnicity were not significantly associated with leukemia-cell ara-CTP level. An Ala119Gly or Pro122Ser non-synonymous polymorphism was present in 1 patient each; the patient heterozygous for Ala119Gly (continuous infusion arm) had the lowest intracellular concentration of ara-CTP (0.012 nmol/2x107 cells; Figure). Both of these patients had relapses, despite the presence of favorable cytogenetic abnormalities [inv16 and t(8;21)]. The 3′UTR 35708 T>C SNP was significantly associated with lower intracellular ara-CTP concentration in patients receiving ara-C as a continuous infusion (35708T allele 0.5±0.3 vs. 35708C allele 0.22±0.17 nmol/2x107 cells; p= 0.04; Figure). These results suggest that genetic polymorphism of DCK influences its activity and expression and may underlie interpatient variability in intracellular ara-CTP concentration, which in turn can influence treatment response. [Display omitted]

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway. We have previously established that Pcyt2 is encoded by a single gene that can be alternatively spliced from an internal exon into two transcripts, designated Pcyt2alpha and Pcyt2beta. Little is currently known about the regulation of Pcyt2. Here, we functionally express both murine Pcyt2 (mPcyt2) transcripts and investigate the roles of the two proteins in the regulation of mPcyt2 activity. We demonstrate that the tagged and purified alpha and beta proteins differ significantly in their kinetic properties. The K(m) of mPcyt2alpha for phosphoethanolamine was 318.4 microM, compared with 140.3 microM for mPcyt2beta. The maximal velocities of the alpha and beta isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/min/mumol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the K(m) of mPcyt2alpha for CTP was 102.0 microM and that of mPcyt2beta was 84.09 microM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2alpha and mPcyt2beta proteins can form both homodimeric and heterodimeric complexes. We show that alternative splicing of the mPcyt2 transcript is ubiquitous but could also be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2alpha/mPcyt2beta mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.

Thyroid Hormone-Induced Alterations in Membrane Structure-Function Relationships: Studies on Kinetic Properties of Rat Kidney Microsomal Na+,K+-ATPase and Lipid/Phospholipid Profiles

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3'-triiodothyronine (T(3)) or combined replacement therapy (T(R)) with T(3 )and thyroxine (T(4)) on the substrate and temperature kinetics properties of Na+,K+-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T(3) treatment, while T(R) treatment caused a decrease. In the Tx and T(3) groups enzyme activity resolved in two kinetic components, while in the T(R) group the enzyme showed allosteric behavior up to 0.5 mM: ATP concentration. The K(m) and V(max) values of both the components decreased in Tx animals without affecting the catalytic efficiency. T(3) treatment caused a significant increase in the V(max) of both the components, with a significant increase in the catalytic efficiency, while the K(m) values were not upregulated. The T(R) regimen lowered the K(m) and V(max) of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components.

Substrate kinetics of DMSP-lyases in axenic cultures and mesocosm populations of Emiliania huxleyi

Most oceanic dimethyl sulphide (DMS) is produced through the enzymatic cleavage of dimethylsulphoniopropionate (DMSP), a process governed by the activity of DMSP-lyases in algae and bacteria. Laboratory and field experiments with the DMS-producing coccolithophorid Emiliania huxleyi have advanced our understanding of algal DMSP lyases. However, despite their central role in the global biogeochemical cycle of sulphur, little is known about the physiological and biochemical properties of this group of isozymes. Here we investigate the apparent substrate kinetic properties of two axenic E. huxleyi cultures (CCMP 373 and CCMP 379) under two light conditions and a mesocosm assemblage dominated by this species. Assays of in vivo and in vitro DMSP-lyase activity (DLA) were used to estimate the kinetic parameters Km and Vmax. Whereas in vivo DLA was not detected in CCMP 379, both laboratory cultures showed high DLA during in vitro tests. The mesocosm population was initially characterised by low substrate affinity (low Vmax and high Km), but shifted to a high affinity community when more dissolved DMSP became available after the collapse of the E. huxleyi bloom. At natural DMSP concentrations, DMS production is probably more affected by changes in Km than Vmax and future studies should include further investigations of the kinetic properties of DMSP-lyases.

A simple quantitative assay for the activity of thymidine kinase 1 in solid tumors

The activity of the pyrimidine salvage pathway enzyme thymidine kinase 1 (TK1) is tightly cell cycle regulated and has been investigated as a prognostic indicator of cancer in a variety of tissues. However, using the in vitro assay of TK1 to rank order a series of unique tumor samples by their TK1 activity can be problematic due to the complex nature of TK1 enzyme substrate kinetics. We present a refined TK1 in vitro assay and method of analysis which address these problems. Extracts were prepared of the resected lung lesions from eight patients and assayed for TK1 activity using an in vitro assay modified to account for nonlinearities in extract protein concentration. A separate extract of exponentially growing A549 human lung carcinoma cells was used as a cross-assay control. In extracts prepared from eight frozen samples of resected human lung lesions, TK1 activity (mean=0.0070+/-0.0077 pmol [(3)H]-TMP/microg protein/minute) was 2 orders of magnitude below that of exponentially growing A549 human lung carcinoma cells (mean=0.1572+/-0.0218 pmol [(3)H]-TMP/microg protein/minute; n=9). TK1 activity was nonlinear with respect to extract protein concentration in both groups, with A549 cell extracts exhibiting evidence of positive cooperativity which could not be explained by the presence of detergents in the cell lysis buffer. Lung tumor extracts demonstrated evidence of negative cooperativity. The modified TK1 assay takes into account these nonlinearities by averaging the results of several complete time-course curves measured over a range of extract protein concentrations. An extract prepared from exponentially growing A549 cells is included in each assay for use as a cross-assay control. We demonstrate that these modifications allow for the accurate rank ordering of TK1 activity in solid tumors.

Pre-operative nutritional status does not alter the metabolic response to major gastrointestinal surgery in patients with oesophageal cancer

Malnutrition is associated with an increased incidence of perioperative morbidity and mortality. To evaluate the effect of malnutrition on the metabolic and inflammatory response to surgery in patients with oesophageal cancer, we studied the effects of oesophagectomy in six patients with major (13.9 (se 1.3) %) weight loss and five patients with minor (0.7 (se 0.6) %) weight loss in the 6 months before to surgery. Rates of appearance (Ra) of glucose, glycerol, leucine and urea were determined by stable isotopically labelled tracer infusion before and after surgery. C-reactive protein was measured as an inflammation marker. BMI was lower in the patients with major weight loss than those with minor weight loss (20.3 (se 0.7) and 24.9 (se 1.5) kg/m2, P = 0.02). With the exception of greater glucose Ra in the major weight loss than minor weight loss subjects (11.1 (se 0.3) v. 9.5 (se 0.3) mumol/kg per min, P = 0.01), there were no differences in substrate kinetics before surgery between groups. Surgery increased glucose Ra, leucine Ra and urea Ra by 41, 24 and 58 %, respectively, in the total group. Changes in substrate kinetics in response to surgery were not different between patients with major and minor weight loss. Surgery increased C-reactive protein concentrations to a comparable extent in both groups. In conclusion, major upper gastrointestinal tract surgery in patients with oesophageal cancer elicits a catabolic response, characterized by increased inflammation, glucose production and protein breakdown. However, this catabolic response does not seem to be influenced by pre-operative nutritional status.