Biomedical subjects

C T Evans

Publications and source records attributed to C T Evans.

At least 19 recordsLinked to original sources

PubMed2004

Management of community-acquired pneumonia in persons with spinal cord injury.

STUDY DESIGN: Retrospective case series. OBJECTIVES: Respiratory disorders are the leading cause of death in persons with spinal cord injury (SCI), but the epidemiology and medical management of pneumonia in persons with chronic SCI is not well characterized. We describe the clinical presentation of persons with SCI with community-acquired pneumonia (CAP), characterize its management and compare practice to recommendations for CAP in the general population. SETTING: Three United States Veterans Affairs Medical Centers with specialized SCI services. METHODS: Chart abstraction was performed for all persons with chronic SCI seen at participating centers for treatment of CAP during a 2-year period. Collected data included presenting signs and symptoms, laboratory and imaging results, initial antibiotic therapy, secretion mobilization techniques, in-patient vs outpatient management, length of stay, and mortality. RESULTS: In all, 41 persons with SCI received treatment for CAP during the study period. A total of 32 (78.0%) patients were admitted for treatment; two (4.8%) required intubation and mechanical ventilation. Initial antibiotic coverage met guideline recommendations for only half of inpatients and infrequently provided adequate antipseudomonal coverage. Microbiologic testing was performed on 26 cases (63.4%) and demonstrated a specific pathogen in only five cases (12.2% of total). Three cases (7.3%) died during treatment for CAP, and 16 (42.1%) of 38 CAP survivors died within a median follow-up of 3 years. CONCLUSION: The majority of chronic SCI patients who present to specialized SCI centers with CAP are admitted for treatment. Short-term mortality is comparable to CAP in the general population.

Adult↗

PubMed2000

Role of citrate synthase in aldosterone-mediated sodium reabsorption.

Aldosterone and other mineralocorticoids increase citrate synthase activity in the kidney and enhance renal sodium reabsorption, but it is unclear whether the increased citrate synthase activity is involved in renal sodium transport. We used the Wistar-Furth rat, an inbred strain found to be deficient in renal citrate synthase activity, as an experimental model to investigate this issue. We confirmed that renal citrate synthase activity from adrenalectomized Wistar-Furth rats was decreased compared with that from control Wistar rats (by 28%). Similarly, urinary citrate excretion was 23% lower in Wistar-Furth rats. Subnormal citrate formation in Wistar-Furth rats could not be accounted for by differences in systemic pH or circulating potassium levels. Because renal citrate synthase activity was reduced in Wistar-Furth rats, we hypothesized that renal sodium excretory responses to mineralocorticoids would be reduced as well. Four-hour sodium excretion after intraperitoneal injection of 5 microg of aldosterone was reduced by 56% in adrenalectomized Wistar rats and by 52% in adrenalectomized Wistar-Furth rats (both P<0.01 compared with vehicle injection). Similarly, the pattern of urinary sodium excretion in response to subcutaneous injections of deoxycorticosterone acetate over a 2-week period was similar in adrenalectomized Wistar and Wistar-Furth rats. In summary, acute and chronic antinatriuretic responses to mineralocorticoids are maintained in Wistar-Furth rats at the level of Wistar rats, despite the marked reduction in citrate synthase activity. These findings are not consistent with an important role for citrate synthase activity in mineralocorticoid-mediated renal sodium transport.

Adrenalectomy↗

PubMed1996

Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties.

We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the kcats and Kms for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the kcat/Km for the individual substrates, OAA and acetyl-CoA. All mutations affected kcat. There was only one mutant, Asp327 Asn, in which the Kms primarily were affected. Most mutations affected both kcat and Km and included the following: His274Gly, His274Arg, Asp375Gly, Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on kcat and Km. The CS mutant Arg401Lys exhibited a modestly improved kcat/Km for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 A resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an "open" conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 A resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an "enolized" version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme.

Amino Acid Sequence↗

PubMed1995

Regulation of eicosanoid synthesis in fibroblasts from inflamed gallbladders.

Gallbladder cell cultures obtained from rabbits subjected to sham or 72 h of bile duct ligation (72 h BDL, cholecystitis model) were incubated with calcium ionophore (A23187), dibutyryl cAMP (cAMP), and phorbol 12,13-diacetate (phorbol) to determine the intracellular signal transduction mechanisms responsible for increased inflamed gallbladder eicosanoid synthesis. Incubation of sham and 72 h BDL cell cultures with A23187 or phorbol significantly increased, whereas cAMP decreased, release of 6-keto-PGF1 alpha, PGE2, thromboxane B2 (measured by enzyme immunoassay) in a dose-related manner. Seventy-two-hour BDL cell cultures contained a specific 2-fold increased level of prostacyclin synthase compared to sham cell cultures which was not altered by preincubation with A23187, phorbol or cAMP. These findings suggest that increased PGI2 release in the sham and inflamed cell cultures following A23187 and phorbol stimulation was mediated in part via the inositol triphosphate pathway and protein kinase C activation and was not associated with altered cyclooxygenase or prostacyclin synthase content.

6-Ketoprostaglandin F1 alpha↗

PubMed1995

Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.

The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates.

Acetyl Coenzyme A↗

PubMed1994

Increased splanchnic prostacyclin synthase and cyclooxygenase content and activity during ischemia is due to new protein synthesis.

BACKGROUND: This study examines the hypothesis that the exaggerated splanchnic release of prostacyclin is due to new synthesis of both cyclooxygenase and prostacyclin synthase (PS) in the ileum muscularis/serosa. METHODS: Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 minutes (shock) or sham shock. The superior mesenteric artery (SMA) was cannulated and removed with its end-organ intestine and perfused in vitro with Krebs-Henseleit buffer with and without cycloheximide (50 micrograms/ml) or indomethacin (20 micrograms/ml). Venous effluent was analyzed for eicosanoids by radioimmunoassay. The SMA, aorta and ileal mucosa, and muscularis/serosa were analyzed for PS and cyclooxygenase content by immunoblot analysis. RESULTS: The sham splanchnic bed released threefold more 6-keto-PGF1 alpha than prostaglandin E2 and thromboxane. Acute ischemia increased splanchnic release of 6-keto-PGF1 alpha threefold compared with sham, which was abolished by cycloheximide or indomethacin treatment. Acute ischemia increased content of PS and cyclooxygenase in the ileal muscularis/serosa twofold and PS in the aorta and SMA by 50%. CONCLUSIONS: Acute ischemia increased release of 6-keto-PGF1 alpha, which was dependent on new protein synthesis. The immunoblot data suggest that the location of the increased enzymes responsible for increased 6-keto-PGF1 alpha release is the ileal muscularis/serosa and in the aorta and SMA.

Animals↗

PubMed1993

[13C]propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization.

The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r. Spectra of cell extracts from wild-type E. coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source. No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate. The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when [2-13C]propionate as opposed to [3-13C]propionate was supplied. Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when [3-13C]propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed. However, in cells supplied with [2-13C]propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets. The unequal and non-identical labelling of glutamate in cells supplied with [2-13C]- as opposed to [3-13C]propionate is consistent with the utilization of propionate by E. coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate. These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle. The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E. coli.

Acetyl Coenzyme A↗

PubMed1993

Acute cholecystitis potentiates bradykinin stimulated fibroblast prostanoid release in the rabbit.

Gallbladder explants from control rabbits and rabbits subjected to bile duct ligation (BDL) for 24 and 72 h (cholecystitis model) were placed in cell culture to determine the source for increased gallbladder prostanoid synthesis during cholecystitis. Cultures from control and 24 h BDL gallbladders grew spindle shaped fibroblasts which did not exhibit increased prostanoid synthesis. 72 h BDL gallbladder cell cultures grew large polygonal shaped cells which appeared to be 'stimulated fibroblasts' by light and electron microscopy and were associated with increased basal and bradykinin stimulated 6-keto-PGF1 alpha release and increased content of prostacyclin synthase when measured by enzyme immunoassay and protein immunoblot analysis respectively. Use of bradykinin antagonists showed that the bradykinin BK2 subtype receptor was the most prominent in the 72 h BDL cell cultures. The 'stimulated fibroblasts' were the source of bradykinin stimulated gallbladder 6-keto-PGF1 alpha synthesis in the inflamed rabbit gallbladder which was mediated by the bradykinin B2 subtype receptor.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation.

Ligation of the common bile duct (BDL) in the male rabbit resulted in increased gall-bladder microsomal total cyclo-oxygenase activity with prostaglandin E2 (PGE2) and 6-oxoprostaglandin F1 alpha [6-oxo-PGF1 alpha, stable metabolite of prostaglandin I2 (PGI2; prostacyclin)] as the major prostanoids synthesized after 24 and 72 h. Kinetic analysis of gallbladder microsomal membrane fractions incubated with increasing levels of [14C]arachidonic acid indicated that BDL for 24 and 72 h did not change substrate affinity (apparent Km) but markedly increased the rate of conversion (apparent Vmax.) suggesting the presence of more total enzyme responsible for synthesis of 6-oxo-PGF1 alpha and PGE2. BDL for 24 and 72 h significantly increased gall-bladder tissue slice basal release of 6-oxo-PGF1 alpha, but not PGE2, when compared with the controls. Gall-bladder slice release of PGE2 was 3-fold less than 6-oxo-PGF1 alpha in the control gall-bladder slices. Immunoblot analysis of 72 h BDL gall-bladder microsomal membrane fractions showed a slight increase in cyclo-oxygenase content and a 5-fold increase in the content of prostacyclin synthase as compared with the control. These data suggest that the BDL-stimulated total gall-bladder cyclo-oxygenase activity was the result of an increase in the level of specific prostaglandin-synthetic enzymes, in particular prostacyclin synthase, and not from a change in enzyme affinity.

Animals↗

PubMed1992

Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes.

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗

PubMed1992

Localization of the citrate synthase (CS) gene to the p12-p13 bands of chromosome 5 in pigs by in situ hybridization.

Citrate synthase (CS) is a key enzyme of the Krebs tricarboxylic acid cycle. A 1.4 kb porcine CS cDNA probe was used to chromosomally localize the CS gene in pigs by in situ hybridization. Two in situ hybridization experiments were conducted. Although the first experiment indicated a distinct signal on the 5p12-p13 bands, a secondary signal was observed on the 13q24-q32 bands. Hence, a second in situ hybridization experiment was conducted at higher stringency. The results demonstrated a consistent signal on the 5p12-p13 bands, and the signal on chromosome 13 was scattered with no prominent secondary peak. The CS gene was therefore assigned to the p12-p13 bands of chromosome 5 in pigs.

Animals↗

PubMed1991

Conformational stability of pig citrate synthase and some active-site mutants.

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.

Animals↗

PubMed1991

Mitochondrial import and processing of rat liver carnitine palmitoyltransferase II defines the amino terminus of the mature protein. Possibility of differential modification of the rat and human isoforms.

[35S]Methionine-labeled porcine heart citrate synthase (used here as a positive control) and rat liver carnitine palmitoyltransferase II (CPT II) were generated by in vitro transcription and translation of their cDNA constructs in appropriate Bluescript plasmids. Each product was imported into rat liver mitochondria in an energy-dependent manner to yield an immunoprecipitable protein of smaller size that comigrated with the corresponding purified enzyme. The size shift occurring with citrate synthase was consistent with the removal of the postulated 27-amino acid leader peptide. To determine the amino terminus of mature CPT II, [35S]methionine- or [3H]leucine-labeled material (after import and processing) was subjected to Edman degradation, followed by counting of the radioactivity released on each cycle. The results established that the precursor targeting peptide was cleaved between leucine 25 and serine 26 in the previously deduced amino acid sequence. Taken in conjunction with the recent report of Finocchiaro et al. (Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 661-665), the present results establish three key points concerning the rat and human forms of CPT II. First, it appears that in both species the initial translation product contains 658 amino acids and, upon mitochondrial import, is reduced in length by 25 residues through cleavage at an identical site. Second, the difference in electrophoretic mobility between the two mature proteins (documented earlier) presumably reflects either anomalous behavior of one of them on polyacrylamide gels or differential covalent modification. Finally, the recent suggestion by Brady et al. (Brady, P. S., Liu, J. S., Park, E. A., Hanson, R. W., and Brady, L. J. (1991) FASEB J. 5, A817) that our CPT II cDNA construct is incomplete in the 5'-coding region is refuted.

Amino Acid Sequence↗

PubMed1990

Mutation of essential catalytic residues in pig citrate synthase.

Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375. The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity. Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum. No activity was detected for Asn375 or Gln375. The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity. The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs. A mechanism is proposed that electrostatically links His274 and Asp375.

Animals↗

PubMed1989

Studies on site directed mutant pig citrate synthases.

The DNAs encoding the non-mutant and mutant forms of pig citrate synthase (PCS) were subcloned into an expression system to determine their synthesis and stability in E. coli gltA- cells that are defective in bacterial citrate synthase. GltA- cells that expressed the non-mutant PCS DNA grew on defined minimal acetate media and produced a constant level of PCS (0.43 U/mg protein). In contrast, when the gltA- cells were transformed with the DNA encoding PCS mutations in His274 or Asp375 the cells did not grow on minimal acetate media. The presence of the mutant PCS proteins in E. coli was confirmed by protein blot and immunoisolation analyses using an antibody specific for porcine heart citrate synthase. The activities of the mutant PCS enzymes were two orders of magnitude less than the non-mutant enzyme in the total cell lysates. The data indicate that the active site amino acids, His274 and Asp375, are essential for the catalysis activity of citrate synthase.

Animals↗

PubMed1989

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme.

We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and alpha-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagensis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.

Acetates↗

PubMed1989

Identification of phenylthiocarbamyl amino acids for compositional analysis by thermospray liquid chromatography/mass spectrometry.

The chromatographic separation of amino acids for compositional analysis of peptides and proteins is commonly performed by reverse-phase high-performance liquid chromatography of amino acid residues that have been derivatized with phenylisothiocyanate. The present report describes an extension of this method, which employs thermospray liquid chromatography/mass spectrometry to confirm the identification of the resulting phenylthiocarbamyl (PTC) amino acids. A standard HPLC separation method has been adapted for use with the thermospray technique, and on-column mass spectra of standard synthetic PTC-amino acids have been acquired. These spectra show characteristic fragmentation patterns not seen in the corresponding cyclic phenylthiohydantoin amino acid derivatives. The LC/MS method has been tested on hydrolysates of bovine serum albumin, porcine insulin, and human placental collagen. In each case, the mass spectra of components eluting with the same retention times as the standard PTC-amino acids are similar to those observed in the standard amino acid mixture. Other components display mass spectra that can be interpreted in terms of known in vivo or in vitro modifications to amino acid side chains in these proteins. The LC/MS method has assisted in the identification of by-products of the derivatization reaction. It has also been applied to a study in which an enzyme, citrate synthase, isolated from porcine heart, was compared to the protein expressed by a recombinant porcine citrate synthase gene in Escherichia coli. The data showed that the recombinant protein lacks a modified residue, trimethyllysine, which is present in the enzyme expressed in mammalian tissues.

Amino Acids↗

PubMed1988

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli.

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.

Acetyl Coenzyme A↗