In the present work, certain biochemical characteristics of the enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase (ACC N-MTase) which is responsible for the malonylation of ...1-aminocyclopropane-1-carboxylate (ACC) in chickpea (Cicer arietinum) are described. Phosphate buffer was the most appropriate buffer with regard to enzyme stability and, therefore, ACC N-MTase was extracted, assayed and purified in the presence of this buffer. ACC N-MTase was partially purified approximately 900-fold from embryonic axes of chick-pea seeds using ammonium sulphate precipitation, hydrophobic interaction and molecular filtration chromatography. By gel filtration chromatography on Superose-12, the molecular mass of the enzyme was estimated to be 54 ± 4 kDa. ACC N-MTase had an optimal pH and temperature of 7.5 and 40 °C, respectively, as well as a Km for ACC and malonyl-CoA of 400 μM and 90 μM, respectively. d-Phenylalanine was a competitive inhibitor of ACC N-MTase with respect to ACC (Ki of 720 μM), whereas co-enzyme A was a competitive product inhibitor with respect to malonyl-CoA (Ki of 300 μM) and a non-competitive inhibitor with respect to ACC (Ki of 600 μM). Under optimal assay conditions, ACC N-MTase was strongly inhibited by (a) divalent Zn2+ > Mg2+ > Cu2+ » Co2+ > (NH4)2+ > Fe2+ and monovalent metal cations (Li+ > Na+ > K+), without activity being detected in the presence of Hg2+, and (b) PCMB or mersalic acid, suggesting that sulphydryl group(s) are involved at the active site of the enzyme.
To assess the significance of increased serum transaminase levels in neonates admitted to a Neonatal Intensive Care Unit and its relationship with blood transfusion.
Follow-up prospective study of ...209 patients; 177 completed follow-up, of whom 129 were transfused and 48 were not; 57 were born after full gestation and 120 were born prematurely. The activity of serum levels of ALT, AST, and GGT was measured monthly up to six months of age, and until six months after the last transfusion. At the end of follow-up, and whenever an increase in serum transaminase levels was detected, the viral agents of hepatitis A, B, C, G, TT, cytomegalovirus, Epstein-Barr, and herpes 1 and 2, and toxoplasma were studied. Viral serology was also carried out in mothers and in donors when children tested positive.
One hundred twenty nine neonates (73%) received 461 U red blood cell transfusions (3.6 +/- 3 U/patient). ALT levels increased in 54 (30.5%) patients, of whom 46 (36%) were transfused and eight (17%) were not (p < 0.05). The independent variables were 'infection by G virus' and 'parenteral nutrition for more than 12 days'; the variable 'transfusion' was close to the limit for statistical significance. Twenty patients (11.3%) had increased serum ALT levels 2.5 times above the normal value: 18 (14%) were transfused and two (4%) were not (p = 0.106). Only the G and TT viruses were related with transfusion; patients remained asymptomatic, although most neonates were chronically infected.
Follow-up showed that increased serum ALT levels are common among severely ill neonates. Blood transfusions are safe concerning most hepatotropic viruses, but transmission of viruses G and TT is possible.
The chemical, physical and biological factors, which can affect the activity of transglutaminase (R-glutaminyl-peptide:amine γ-glutamyl-transferase, EC 2.3.2.13) in chloroplasts (ChlTGase) when ...photosynthesis is active, were assayed in chloroplasts isolated from the leaves of
Helianthus tuberosus. Chloroplasts were incubated with putrescine (PU) in the presence of light to monitor the transglutaminase-catalysed incorporation of this polyamine into endogenous proteins. The enzyme was identified using a monoclonal antibody raised against the active site sequence of TGase K and was found to contain a thiol group, which can be slightly activated by Ca
2+ and severely inhibited by EGTA. Mg
2+ had a slight inhibitory effect. The enzymic activity, monitored by the isolation of glutamyl-putrescine, while already detectable above pH 7 was found to increase sharply from pH 8.0 to 9.5, with an optimal temperature of 45 °C. A hyperbolic curve was observed when the activity was measured as a function of the putrescine concentration, the apparent
K
m being 1 mM. A biphasic relationship was obtained between the TGase activity and the concentration of the substrate (endogenous proteins) as well as the time of assay. The reaction products of the TGase assay, carried out at three pH values, were analysed for the presence of γ-glutamyl-putrescine; mono- and bis-derivatives were detected, showing that most of the modifications of Chl proteins are catalysed by the enzyme. Due to the stimulatory effect that proteases have on some animal TGases, protease inhibitors were also tested and found to reduce the post-translational modification of the substrates.
For a deeper understanding of the germination of chick-pea (Cicer arietinum) seeds, which is dependent upon ethylene synthesis, a crude extract containing authentic ACC oxidase (ACCO) activity was ...isolated in soluble form from the embryonic axes of seeds germinated for 24 h. Under our optimal assay conditions (200 mM HEPES at pH 7.0, 4 μM FeSO4, 6 mM Na-ascorbate, 1 mM ACC, 20% O2, 3% CO2, and 10% glycerol) this enzyme was 5-fold more active than under the conditions we used initially in the present work. The enzyme has the following Km: 28 μM for ACC (approximately 4-fold less than in vivo), 1.2% for O2 (in the presence of an optimal CO2 concentration of 3%), and 1% for CO2 in the presence of O2 (20%). The enzyme is inhibited by phenanthroline (PNT) (specific chelating agent of ferrous ion), and competitively inhibited (Ki =0.5 mM) by 2-aminoisobutyric acid (AIB), and the enzymatic activity was not detectable in the absence of CO2. Under optimal assay conditions, the enzyme has two optimum temperatures (28 °C and 35 °C) and is inhibited by divalent metal cations (Zn2+ ≥ Co2+ > Ni2+ >Cu2+ >Mn2+ >Mg2+) and by salicylic acid, propylgallate, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and Na-benzoate. The in vitro ACCO activity which we recovered in soluble form is equivalent to approximately 80-85% of the apparent activity evaluated in vivo.
This study was undertaken to determine the prevalence, epidemiology, and mother-child repercussions of increased alanine-aminotransferase levels from week 16 of pregnancy.
A longitudinal ...observational study of 381 pregnant women. The cause of increased alanine-aminotransferase levels during pregnancy and repercussions on the neonate were studied in 283 cases. Statistical analysis was performed with Mann-Whitney test, χ
2 test, or the Fisher exact test.
The mean age of the mothers was 29.9 ± 4.8 years. Twenty-five percent presented increased gamma-glutamyl-transpeptidase, alkaline phophatase, and dehydrogenase lactate from week 32. Increased alanine-aminotransferase was observed in 7.4% (95% CI, 5.00%-10.57%) of cases. Clinical disorders were light, transitory, and with no apparent cause, except for 1 hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, 3 preeclampsias, and 1 gravidic cholestasis. No statistically significant differences were observed in the group of mother-child with alanine-aminotransferase normal or increased.
Most increases in alanine-aminotransferase from week 16 of pregnancy are transitory, non-specific, and have no repercussions on mother or child.
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