Delta-Aminolevulinic acid in urine (screening method)

Various methods for determining urinary delta-aminolevulinic acid (ALA) have been devised by many investigators since 1956. This paper introduces a history of the methodology in the determination of urinary ALA and application of some methods. These methods can be divided into two groups; one group consists of colorimetric methods based on the color reaction of ALA-pyrrole with Ehrlich's reagent, and the other group consists of fluorometric methods, based on the fluorescence derivatization of ALA and its separation by HPLC. Colorimetric methods are convenient and inexpensive, while these are less specific. On the other hand, the fluorometric HPLC methods are highly sensitive and specific, while these are expensive because of the high cost of the instruments.     

Switching the amino acid specificity of an aminoacyl-tRNA synthetase

The accuracy of protein synthesis essentially rests on aminoacyl-tRNA synthetases that ensure the correct attachment of an amino acid to the cognate tRNA molecule. The selection of the amino acid substrate involves a recognition stage generally followed by a proofreading reaction. Therefore, to change the amino acid specificity of a synthetase in the aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to accommodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis designates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by duplication of the gene for glutamyl-tRNA synthetase (GluRS). By introducing GluRS-specific features into the Rossmann dinucleotide-binding domain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data suggest that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.     

Cytochrome p-450 heme and the regulation of delta-aminolevulinic acid synthetase in the liver

Twenty-five human gastric and 11 human colonic adenocarcinomas were analysed for their ganglioside pattern and for their content of lipid-bound and protein-bound neuraminic acid. In most carcinomas the content of both lipid-bound and protein-bound neuraminic acid was increased by an average of four- and two-fold, respectively. The ganglioside pattern of all the carcinomas resembled that of normal tissue. In six gastric carcinomas the content of lipid-bound neuraminic acid and the ratio of lipid-bound neuraminic acid to protein-bound neuraminic acid (L/P ratio) were lower than those of normal gastric mucosa. These carcinomas were significantly larger than the rest of the tumours.     

Organization of the genes encoding p-aminobenzoic acid synthetase from Streptomyces lividans 1326

The selective 5HT3 antagonist tropisetron was studied in 91 outpatients meeting DSM-III criteria for Generalized Anxiety Disorder. Following a placebo washout period of up to 1 week, one of three active treatments (tropisetron 0.5 mg, 5 mg, or 25 mg daily) or placebo was given for a further 3 weeks. After 7 days treatment termination rates due to inefficacy showed a statistically significant dose-related therapeutic effect of tropisetron. Similar effects were seen on the Hopkins Symptom Check List total score and the Global Impression Scale. The Hamilton Anxiety Scale showed a similar trend which, however, failed to reach statistical significance. At day 21 tropisetron showed significant dose-dependent effects on all anxiety-related outcome measures. The incidence of adverse events was low and the severity generally mild. Most frequent complaints were headache, nausea, constipation and nervousness. Laboratory tests and physical examination performed at baseline and study end showed no significant treatment effects.     

Mycobacterium smegmatis fatty acid synthetase. Polysaccharide stimulation of the rate-limiting step

An initial activity burst lasting 5 to 10 s is observed for both de novo synthesis with acetyl-CoA as primer and for elongation of palmitoyl-CoA catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis. After the initial burst, synthetase activity slows at least 6-fold to the steady state rate. The size of the initial burst is proportional to the amount of synthetase protein and corresponds to the synthesis of a small number C three to five) of C24 or C26 acyl chains per mol of enzyme. During the initial burst, C24, C26 acyl enzyme is formed and can be isolated by ammonium sulfate precipitation. On incubation with CoA, enzyme-bound acyl chains undergo transacylation to form the corresponding CoA derivatives. Diffusion of C24-CoA and C26-CoA from the enzyme is slow and rate-limiting for overall fatty acid synthesis. Mycobacterial polysaccharides markedly accelerate this rate-determining step but bovine serum albumin does not. This facilitation of product diffusion accounts for the large stimulation of de novo synthesis and of elongation of mycobacterial polysaccharide. It is also shown that the high apparent Km for acetyl-CoA (approximately 400 micrometer) in the steady state reflects the substrate concentration required to shift the product pattern in favor of shorter chain fatty acids (C16,C18). These conditions circumvent the slow, rate-limiting diffusion of C24-CoA and C26-CoA.     

Analysis of amino acid sequences of Rhodobacter sphaeroides glutamine synthetase

A comparative analysis of the amino acid sequence of glutamine synthetase (GS) of the photosynthetic purple bacterium Rhodobacter sphaeroides revealed that the enzyme is typical for first type procaryotic GSs and structurally resembles GSs of enteric bacteria. The data obtained indicate that the complex phenotype of purple bacterial mutants at the glnA gene coding for GS may be conditioned by specific regulation of nitrogen metabolism in bacterial cells rather than by structural-and-functional peculiarities of GS.     

Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12

GDP-L-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.     

ATP-dependent inactivation of Escherichia coli gamma-glutamylcysteine synthetase by L-glutamic acid gamma-monohydroxamate

Incubation of Escherichia coli gamma-glutamylcysteine synthetase with L-glutamic acid gamma-monohydroxamate and ATP caused slow but irreversible inhibition of the enzyme, and more than 90% activity was lost in three days. The enzyme was not inactivated when ATP was absent or L-aspartic acid beta-monohydroxamate was substituted for L-glutamic acid gamma-monohydroxamate, suggesting that the inactivation process reflected a mechanism-based reaction of L-glutamic acid gamma-monohydroxamate and ATP.     

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.     

Regulation of phosphoenolpyruvate carboxykinase and fatty acid synthetase in brown fat of suckling rats

Infant rats were injected with prednisolone (0.5-5 mg/100 g wt). This caused phosphoenolpyruvate carboxykinase (PEPCK) activity to rise in liver and to decrease in brown fat. Fatty acid synthetase (FAS) activity remained unchanged in liver but increased in brown fat. A single injection of prednisolone caused hepatic PEPCK activity to remain elevated for at least 7 days. Brown fat FAS also remained high for that period. However, brown fat PEPK activity returned to normal on the third day after the injection. A single injection of prednisolone or cortisone to 5-day-old rats caused a transient elevation of the blood level of insulin and a prolonged decrease in that of growth hormone. No effect on the level of glucagon was noted. Injections of insulin had effects similar to those of prednisolone, i.e. a rise in hepatic and a fall in brown fat PEPCK. Using antibodies prepared to hepatic PEPCK it was shown that the observed changes were due to changes in the rate of synthesis of the enzyme. Using actinomycin D indirect evidence was obtained that changes in FAS activity of brown fat were also due to changes in the synthetic rate.     

Mycobacterium smegmatis fatty acid synthetase. Long chain transacylase chain length specificity

Long chain transacylase activity, acyl-CoA + enzyme in equilibrium acyl-enzyme + CoA, catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis was measured by exchange of radioactive coenzyme A into even numbered fatty acyl-CoA substrates 14 to 24 carbon atoms long. This transacylase activity decreases sharply with increasing chain length. It is suggested that C24 transacylation may be rate-limiting in de novo fatty acid synthesis catalyzed by the myocobacterial system. Mycobacterial polysaccharides stimulate the rate of transacylation, and this enhancement becomes more marked as the chain length of the substrate increases. The magnitude of the effect is similar to polysaccharide stimulation of overall synthetase activity. It is therefore proposed that terminal transacylation is the specific and perhaps only partial reaction catalyzed by the M. smegmatis fatty acid synthetase which is facilitated by polysaccharide. The product distribution of the synthetase is distinctly bimodal, with peaks for acyl chains 16 and 24 carbon atoms long. A scheme based on nonoverlapping unimodal chain length specificities for the rates of two activities, elongation and terminal transacylation, is offered to explain this bimodal distribution.     

Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase

The present studies were designed to evaluate the developmental toxicity of chlorpropham in mice. The first study was conducted to determine administration time, and the second study was designed to evaluate dose-response effects. Chlorpropham was administered to pregnant mice by gavage on Days 8, 8.3, 9, 9.3, 10, and 11 of gestation at a level of 3000 mg/kg bw, and the females were killed on Day 18 of gestation. The administration on Day 8.3 of gestation induced the highest percentage of external malformations with brachyury occurring among more litters than in other groups. Chlorpropham was administered to pregnant mice by gavage at a level of 0 (control), 750, 1500, and 3000 mg/kg bw on Day 8.3 of gestation, and the females were killed on Day 18 of gestation. The total resorption rate was significantly increased in the 3000 mg/kg bw group. The average fetal body weight of each sex was significantly reduced in the 3000 mg/kg treatment group. The total incidence of external malformations was significantly increased in the two highest dose groups in a dose-related manner. Again brachyury was significantly increased in the 3000 mg/kg bw group.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5--8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

Metabolites influence control of lysine transfer ribonucleic acid synthetase formation in Escherichia coli K-12

A mutant of E. coli K-12 has been isolated which has only 1-3% of the wild-type lysyl-tRNA synthetase activity [L-lysine:tRNA ligase (AMP forming), EC 6.1.1.6]. Additions of 20 mM L-alanine or 6 mM leucine dipeptides to the culture medium can restore the activity of lysyl-tRNA synthetase in the mutant strain to the wild-type level. Experiments on the in vivo charging of lysine tRNA in the mutant show that in the absence of the metabolites lysine tRNA is charged 15-23%. Upon the addition of 3 mM L-leucyl-L-alanine to the medium the lysyl tRNA synthetase activity increases 25-fold and the in vivo charging of lysine tRNA returns to the wild-type level. Experiments with antibody against lysyl-tRNA synthetase show that the stimulation of lysyl-tRNA synthetase activity by the metabolites is the result of new protein synthesis.     

Fatty acid synthetase from the Harderian gland of guinea pig: biosynthesis of methyl-branched fatty acids

Fatty acid synthetase was isolated from guinea pig Harderian gland. This enzyme complex exhibited a unique character as compared with the fatty acid synthetase from the liver of the same animal. The former enzyme produced many odd-numbered and methyl-branched fatty acids in the presence of methylmalonyl-CoA. These fatty acids are characteristic components of the lipid secreted from this gland. The chemical structure of this lipid has been identified as 1-O-alkyl-2,3-diacylglycerol by previous work from this laboratory (Yamazaki, T., Seyama, Y., Otsuka, H., Ogawa, H., & Yamakawa, T. (1981) J. Biochem. 89, 683-691). Apparent Km values (5 X 10(-6) M) for acetyl-CoA and propionyl-CoA were the same, but the Vmax for propionyl-CoA was much higher than that for acetyl-CoA. The pI value of the fatty acid synthetase from Harderian gland was 5.3, and the molecular weight of the enzyme was 9 X 10(5) daltons. The beta-ketoacyl reductase had pro-S stereospecificity and the enoly reductase had pro-R stereospecificity for NADPH. The results presented in this paper indicate that the fatty acid synthetase from guinea pig Harderian gland can produce a set of fatty acids needed for the synthesis of the lipid secreted from this gland, and that the fatty acid synthetase has a characteristic organ specificity.     

Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.