N-Acetylglutamate synthetase: enzyme assay in human liver

In the context of diagnostic procedures for congenital hyperammonaemias a methods is described for the determination of N-acetylglutamate synthetase in human liver tissue homogenates. The method uses [14C-U] glutamate and acetyl CoA as substrates. The reaction product, N-acetylglutamate is separated from the substrate L-glutamate by chromatography on Extrelut. In a subsequent step on ITLC-SG ready plates N-acetylglutamate is separated from other labeled metabolites such as Krebs cycle intermediates. The recovery of N-acetylglutamate was 97.8%. The precision within run and between days was 8.5% (CV) and 9.6% (CV) respectively. Reference values were established for adult human liver.     

Chitobiase, a new reporter enzyme

Traditional inferential statistics require that hypotheses be evaluated at only 1 sample size. That is, researchers must choose how many participants will be included in a study before conducting analyses; they are not allowed to add data if initial results are not significant. This requirement forces researchers to choose among including more participants than necessary, risking inconclusive results, or violating the requirement by adding participants. This study presents a more flexible approach, called data monitoring, that allows repeating an analysis as the sample increases. First, the cost of the uncorrected data monitoring that researchers sometimes do is estimated. Second, the correction that is needed to allow data monitoring while holding an overall alpha at a desired level is estimated. Third, the power of data monitoring is compared with traditional approaches. This study also provides an example of the use of data monitoring. At least in some circumstances, data monitoring can reduce Type II error or the number of participants needed without sacrificing Type I error.     

Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway

The work presents the results of researches of binding and degradation of 125I-Insulin by erythrocyte receptors in the patients with essential hypertension and healthy patients after glucose intake. In order to obtain full representation of the pattern of changes the serum IRI and glucose concentrations were assayed. Binding and degradation of 125I-Insulin by erythrocyte receptors were determined with the method described by Gambhir (1977), modified by the authors. The modification consisted in usage of constant concentrations of iodized insulin (0.9 pg/0.1 ml) and bovine insulin (2.4 I.U./0.1 ml). Before administration of glucose and in 30, 60 and 120 minutes after, venous blood was collected from ulnar vein. All examined persons were in sitting position during the trial of glucose intake. Obtained results show, that blood insulin level in the patients with essential hypertension is statistically significantly higher than in healthy persons of similar anthropometric characteristics. Binding of 125I-Insulin to erythrocyte receptors in fasting state is statistically significantly lower. Degradation after glucose intake in the patients shows decreasing tendency, while in healthy persons--growing tendency.     

Aspartate analysis in formulations using a new enzyme sensor

A biosensor has been developed for the purpose of directly analysing aspartate in pharmaceutical formulations and aspartame in sweeteners. This biosensor consists of an ammonia-sensitive gas-diffusion electrode and the enzyme L-aspartase immobilized by means of polyazetidine on a dialysis membrane.     

Alterations in cholesterol sulfate and its biosynthetic enzyme during multistage carcinogenesis in mouse skin

A novel series of nonpeptide angiotensin II antagonists containing the acrylamide group at the 4-position of the imidazole ring was synthesized and their antagonistic activity was examined by functional assay in rabbit aorta. The acrylamide group was selected as a large lipophilic surrogate for the chloro group of EXP3174. A structure-activity relationship study of the acrylamide moiety has shown that substitution at the 4-position with the N-methyl-3,3-dimethylacrylamide group resulted in the optimal compound, 2-butyl-4-[(3,3-dimethylacryloyl)methyl-amino]-1-[[2'-(1H-tetra zol-5- yl)biphenyl-4-yl]methyl]-1H-imidazole-5-carboxylic acid (1), which was superior to EXP3174 in vitro. Since 1 showed only poor activity against angiotensin II-induced pressor response in rats after oral administration, the carboxylic acid function of 1 was converted into prodrug esters (13). Among these, the 1-[(ethoxycarbonyl)oxy]ethyl ester (13a) showed the most potent and longest-lasting activity when given orally to rats. When administered orally to conscious furosemide-treated dogs, 13a showed an approximately 3-fold increased hypotensive activity in comparison with DuP 753. These data suggest that 13a may be an useful agent for the treatment of angiotensin II-dependent diseases, such as hypertension.     

Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit

We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exceptionally large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain of two repeats of an approximately 90-aa sequence appended to its C-terminal end. While extracts of Escherichia coli do not aminoacrylate mammalian tRNA with isoleucine, expression of the cloned human gene in E. coli results in charging of the mammalian tRNA substrate. The appended third domain is dispensable for detection of this aminoacylation activity and may be needed for assembly of a multisynthetase complex in mammalian cells. Alignment of the sequences of the remaining two domains shared by isoleucyl-tRNA synthetases from E. coli to human reveals a much greater selective pressure on the domain needed for tRNA acceptor helix interactions and catalysis than on the domain needed for interactions with the anticodon. This result may have implications for the historical development of an operational RNA code for amino acids.     

ACV synthetase: expression of amino acid activating domains of the Penicillium chrysogenum enzyme in Aspergillus nidulans

A 51-year-old woman developed an acute onset of renal dysfunction accompanied by rash, lumbar pain, arthralgias, fever, eosinophiluria, and an elevated serum creatinine after 6 days of intravenous piperacillin-tazobactam therapy. On discontinuing piperacillin-tazobactam and after a 21-day course of prednisone, the patient's constitutional symptoms dissipated and her renal function returned to baseline. Acute interstitial nephritis has been reported as an adverse effect of many drugs, including antibiotics, but not, to our knowledge, after piperacillin-tazobactam. The time course of events suggested that piperacillin-tazobactam was the cause of acute interstitial nephritis in this patient.     

Enzyme-linked immunosorbent assay of carbamoylphosphate synthetase I: plasma enzyme in rat experimental hepatitis and its clearance

Carbamoylphosphate synthetase I (CPS I), a urea cycle enzyme, is located almost exclusively in the mitochondria of hepatocytes. The enzyme is unique in that it constitutes about 2-6% of total liver protein and is composed of a large subunit of 160 kD. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for measurement of the enzyme in plasma using an antibody against the rat enzyme. In galactosamine-induced rat acute hepatitis, plasma concentration of CPS I that was 1-2 micrograms/ml blood before the treatment, increased up to 125 micrograms/ml blood in 24 h after the treatment and decreased to a near control level in 72 h. Plasma concentration of ornithine carbamoyl-transferase (OCT), another urea cycle enzyme, reached a maximum in 24 h and then decreased a little more rapidly than that of CPS I. On the other hand, alanine aminotransferase activity reached a maximum in 36 h and decreased to a normal level in 96 h. In immunoblot analysis, the native CPS I polypeptide of 160 kD and its fragments of 140 and 125 kD were detected 24-48 h after the treatment. When purified rat CPS I and bovine OCT were injected intravenously into rats, the enzymes disappeared from blood roughly exponentially with apparent half-lives of about 67 and 18 min, respectively. Development of an ELISA for human CPS I and determination of the serum enzyme in various liver diseases remain to be performed.     

Carbamoyl phosphate synthetase: a tunnel runs through it

BACKGROUND: Sensitivity to specific allergens and increased sensitivity to common spasmogens are characteristic features of allergic asthma and are also features demonstrated by tissues passively sensitized with serum from atopic donors, displaying high levels of IgE. It is evident that the specific response to allergen is related to circulating levels of allergen-specific IgE, but it is unclear whether the histamine hypersensitivity is also related to this immunoglobulin. OBJECTIVE: The objective was to deplete IgE in the serum of a donor with high levels of total and allergen-specific IgE and compare specific-allergen sensitivity and sensitivity to histamine in tissues passively sensitized with either the whole serum or the IgE-depleted serum. METHODS: Serum from a Dermatophagoides farinae-sensitive asthmatic (total IgE = 1047 U/mL, D. farinae-specific IgE > 17.5 U/mL) was subjected to an immunomagnetic separation technique to reduce the levels of IgE (total and specific) to below 10 U/mL. Bronchial tissue from six non-atopic donors was then passively sensitized overnight with either the whole serum or IgE-depleted serum and D. farinae and histamine sensitivity were evaluated the next day using standard organ bath techniques. RESULTS: Passive sensitization with the whole serum resulted in the development of sensitivity to D. farinae and increased sensitivity to histamine (750+/-169 mg contraction to 10 U/mL D. farinae, histamine pEC50 5.64+/-0.16 and max. 813+/-109 mg in sensitized vs 37+/-34 mg contraction to 10 U/mL D. farinae histamine pEC50 5.05+/-0.23 and max. 490+/-84 in non-sensitized tissues, P>0.05). Incubation with IgE-depleted serum still produced histamine hypersensitivity (histamine pEC50 5.57+/-0.16 and max. 737+/-70 mg P>0.05), but no significant response to allergen was detected (20+/-13 mg contraction to 10 U/mL D. farinae). CONCLUSION: These results demonstrate, that increased reactivity to histamine and airway contraction to allergen induced by passive sensitization, occur through independent mechanisms and that, unlike allergen-sensitivity, histamine hypersensitivity is caused by a serum factor other than IgE.     

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.     

Carbonic anhydrase: a multigene-multifunctional enzyme

Carbonic anhydrase is a zinc metalloenzyme that catalyzes the simple interconversion between carbon dioxide (CO2) and bicarbonate (HCO3-). Seven genes encode the CA isozymes in vertebrates. They are single chain peptides termed CAI-VII. One CA isozyme is present in teleost fish. Three isozymes clearly appear together in birds. All seven types appear in mammals. Despite the great similarity among these isozymes, they present strong differences with respect to their kinetic properties. Many physiological and biochemical processes are related to the activity of CA isozymes.     

A new chromogenic substrate for angiotensin-converting enzyme

The compound para-nitrobenzyloxycarbonylglycyl-(S-4-nitrobenzo-2-oxa- 1,3-diazole)-L-cysteinylglycine [NO2ZGly(S-NBD)CysGly] with an absorption maximum at 423 nm is readily hydrolyzed by angiotensin-converting enzyme (EC 3.4.15.1. peptidlyldipeptide hydrolase) to yield the S-benzfurazan derivative of cysteinylglycine. An internal S-->N shift occurs immediately to yield the N-benzfurazan derivative which in turn reacts with the sulfhydryl reagent 4,4'-dithiodipyridine to produce the mixed disulfide with an intense absorption at 461 nm. The maximum difference in molar absorptivity of 13,000 M-1 cm-1 occurs at 470 nm.     

Structure of D-amino acid oxidase: new insights from an old enzyme

D-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of D-amino acid dehydrogenation, with significant implications for the general understanding of flavin-dependent catalysis.