Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.6.1.1 (aspartate aminotransferase)
 21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An adult woman diagnosed with cerebral thrombosis following a caesarean section was found to have severely prolonged thrombin and reptilase times. Five other family members also had prolonged, but variable, thrombin and reptilase times. Analysis of purified fibrinogen on reducing SDS-PAGE revealed an additional band, in all family members, which migrated immediately below the normal B beta band. Western blotting indicated that this band was a gamma chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide side chain. Partial acid hydrolysis localized the new oligosaccharide to the C-terminus of the gamma chain. Amplification of this region by PCR and subsequent DNA sequencing demonstrated a single base substitution altering the normal 380 Lys (AAG) codon to Asn (AAT), producing a new Asn-Lys-Thr glycosylation site. The propositus and one other family member were homozygous for this mutation but the remaining four family members were heterozygous. The polymerization of purified fibrin monomers from the propositus was grossly abnormal; however, the polymerization curve was almost normalized by the removal of terminal sialic acid residues. This suggests that the polymerization defect was primarily caused by additional negatively charged sialic acid residues present on the new oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site remained unaffected by the bulky carbohydrate side chain or negatively charged sialic acid residues.
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PMID:Fibrinogen Kaiserslautern (gamma 380 Lys to Asn): a new glycosylated fibrinogen variant with delayed polymerization. 940 Oct 66

A new AAT allele (PI Zbristol) has been discovered in a woman with an obstetric history of three perinatal deaths from fulminant liver disease and no living offspring. She and her father were both PI M1Zbristol heterozygotes. The Zbristol protein is active as a proteinase inhibitor but appeared to be deficient in the plasma to about the same degree as the S protein in MS heterozygotes. It focuses on the basic side of Z and lacks the normal pattern of secondary isoforms associated with the commonly occurring AAT variants and migrates faster than normal on an SDS electrophoresis gel. The Zbristol mutation was found to be a C to T transition at codon 85 changing ACG (Thr) to ATG (Met). This disrupts the N-glycosylation site starting at Asn 83 preventing glycosylation at residue 83 in the PI Zbristol protein and explains the protein isoelectric focusing and SDS gel electrophoresis results. An analysis of haplotypes in the propositus and her father indicated that the Zbristol mutation occurred on the common M1(Val 213) genetic background. The new mutation also led to the generation of an NlaIII restriction endonuclease recognition site. Cell lines from two offspring tested for the presence of this NlaIII site revealed that one had the variant and the other did not. Thus, the relationship between Zbristol and fulminant liver disease in the offspring is unclear.
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PMID:A new alpha 1-antitrypsin mutation, Thr-Met 85, (PI Zbristol) associated with novel electrophoretic properties. 945

Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS-PAGE. AST-RB1 efficiently catalyzed N-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3, 6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.
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PMID:Purification and characterization of two amine N-sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from liver cytosols of male rabbits. 998 35

Carbohydrate-derived aldehydes cause irreversible loss of protein function via glycation. We previously observed that glyceraldehyde 3-phosphate (Glyc3P) abolishes the enzyme activity of cardiac aspartate aminotransferase (cAAT). We also examined the protective effects of carnosine against Glyc3P-induced loss of enzyme activity. The present study looked at carnosine's prevention of Glyc3P-induced change in protein structure. Purified cAAT (2 mg protein/mL) was incubated with various concentrations of carnosine (1-20 mM) in the presence of Glyc3P (500 microM) for 4 days at 37 degrees C. Following incubation, samples were analyzed by SDS-polyacrylamide gel electrophoresis. Carnosine showed prevention of protein modification at carnosine-to-Glyc3P ratios of 10:1 or greater. There was a progressive loss of the unmodified cAAT protein band as Glyc3P concentration was increased. Additionally, the gel position of the Glyc3P-modified cAAT protein varied over time. The apparent molecular weight (MWapp) of the Glyc3P-modified cAAT protein that formed after 1 day at 37 degrees C (500 microM) was greater than its MWapp after 2 days, suggesting that a chemical rearrangement of the initial adduct occurs. These observations support the hypothesis that carnosine is an antiglycation agent and that its mechanism of action involves prevention of protein modification.
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PMID:Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase. 1078

Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT- 2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.
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PMID:Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L. cv Estoril. 1229 33

Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.
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PMID:Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita. 1499 Feb 16

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.
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PMID:Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes. 1615 36

Three oils were tested for their molluscicidal activity, Caple-2, Kemasol and Super-max. Super-max had the strongest toxic effect on B. alexandrina and other snail species. Its LC50 was 0.53 ppm, meanwhile LC50 of Kemasol 3.2 ppm and 4.21 ppm for Caple 2. The LC50 & LC90 of the oils were lower in Lymneae natalensis as compared to B. alexandrina. The LC50 & LC90 of the oils against non-target snails (Physa acuta, Helisoma duryi, Planorbis planorbis and Melanoides tuberculata) were higher as compared to B. alexandrina. Hatchability of snails' eggs exposed to Super-max (3.0 & 5.0 ppm) was stopped completely and l.0 ppm showed the lower percent of egg hatchability 22.7 %. Caple 2 and Kemasol did not affect eggs hatchability. Supermax had the strongest harmful effect on both miracidia and cercariae of S. mansoni. 100% mortality values were obtained for both larval stages after 8 & 9 minutes respectively when maintained at LC50. 100% mortality of miracidia occurred after 35 & 155 minutes when maintained at LC50 of Kemasol & Caple 2 respectively. The infection rate of B. alexandrina with S. mansoni miracidia was greatly reduced by the sublethal concentrations of the oils. The reduction of infection rate was higher in snails treated with Supermax (42.9%). A highly significant reduction of total cercarial production per snail was in the experimental groups as compared with controls. The prepatent period of treated snails was prolonged compared to control. Moreover, Total protein content and enzyme activities of snails treated with LC10 of oils showed a significant reduction as compared with control in haemolymyph. There was an increase of protein contents in the tissue. AlkP enzyme activity was slightly increased in haemolymph of experimental groups than controls and was significantly higher in the tissues as compared to control. ALT enzyme activity in haemolymph of experimental groups was higher than control, but lower in tissue. AST enzyme activity was higher in haemolymph and tissue of experimental groups than controls. The SDS-PAGE pattern of tissue soluble proteins extracted from treated B. alexandrina and controls showed different oils effects on the synthesis of protein within snails yielded a complex pattern of polypeptides ranging in molecular weight between 13.775 to 156.7 kDa. Many bands were present in treated snails. At least, one band was detected for snails treated with each of the oils and not in controls. The difference in the similarity indices between treatment and control; for Kemasol was 0.86 & 0.64, for Caple 2 was 0.61 & 0.55 and for Supermax was 0.64 & 0.86. LC25 of Supermax did not cause any mortality to Daphnia after 6 hr. But, LC50 & LC90 caused lower mortality after 6 hr. Kemasol caused 100% mortality after 4 hr at LC50 and 2 hr in LC90. Caple 2 caused 50% mortality of Daphnia after 5 hr at LC25 &100% mortality after 30 minutes in LC50 & LC90.
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PMID:Effect of three dormant oils on schistosomiasis and fascioliasis vector snails and its relation with some non-target snails. 1715 97

Obesity is considered to be epidemic worldwide. Stopping further progression interdisciplinary, outpatient intervention therapy programs for obese children have become increasingly important. FITOC (Freiburg Intervention Trial for Obese Children) consists of a combination of organized sports, behavioral therapy and nutritional advice. The effectiveness of the therapy is determined on the basis of anthropometrical and physical performance data. The purpose of this report is to give a differentiated view of the motor abilities of obese children and to describe changes in the course of the therapy program FITOC. Data were collected on n = 49 obese children (BMI > 97th percentile) aged 8 - 12 in a pretest at the beginning and posttest at the end of the intensive phase of the therapy. These data were compared with an age-matched German reference group. Besides the General Sports-Motor Test (Allgemeiner Sportmotorischer Test [AST]), the BMI-SDS values, the body fat mass (FM %) and the aerobic capacity (Watt/kg body weight) were recorded. In the pretest, the running exercise results and the aerobic capacity checked ranged significantly below the values of the reference group. The performance in the coordinative tests of the AST was differentiated. The medicine-ball toss was significantly above average of the reference group. In the posttest, the BMI-SDS values and the body fat mass (% FM) decreased (p < 0.001) and the aerobic capacity improved (p < 0.001). Performance in all motor abilities tests improved and the difference between the strength of the obese children and the strength of the reference group decreased. This study demonstrates that in obese children weight-bearing activities are below average but not all motor abilities.
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PMID:Motor abilities and aerobic fitness of obese children. 1749 79

The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 10(12) PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 microg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at doses of 1 x 10(8) or 1 x 10(7) PIBs/rat, respectively) appeared to be healthy and showed increased body weight at 21 days posttreatment. The effect of virus administration on hematological, serum biochemical, and histopathological parameters were determined. Slight to moderate differences were observed in most of the hematological parameters. Specifically, serum proteins were decreased markedly in female rats treated orally with SlNPV, and in male rats injected with AcAaIT. SDS-PAGE analysis also showed some changes in serum protein profiles. No marked changes in acetylcholine esterase (AChE) activity were found. Changes in serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, creatinin, and urea were also observed. Immunohistochemical observation of tissues from stomach, intestine, liver, kidney, brain, spleen, and lung also showed slight changes. Fish (Tilapia nilotica) were also exposed to AcAaIT, AcMNPV or SlNPV by incorporating each of the viruses into diet (1 x 10(9) PIBs/group). No mortality was found in treated or untreated fish during the experimental period (28 days). Macrophage phagocytic activity of fish head kidney cells increased with time, reaching maximum values at 180 min for both treated and control fish.
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PMID:Biosafety of recombinant and wild type nucleopolyhedroviruses as bioinsecticides. 1761 74


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