Co-purification of alanine-glyoxylate aminotransferase with 2-aminobutyrate aminotransferase in rat kidney.

Alanine-glyoxylate aminotransferase and 2-aminobutyrate aminotransferase were co-purified from rat kidney to a single protein (about 500-fold purified from the homogenate). The activity ratios of alanine-glyoxylate aminotransferase to 2-aminobutyrate aminotransferase were constant during co-purification steps suggesting the 2-aminobutyrate aminotransferase activity was catalysed by only alanine-glyoxylate aminotransferase. The molecular weight of the enzyme was estimated to be approx. 213 000, 220 000 and 236 000 by analytical ultracentrifugation, Sephadex G-150 gel filtration and sucrose density gradient centrifugation, respectively. From the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the enzyme consisted of four apparently similar subunits having a molecular weight of approx. 56 000. The enzyme was almost specific to L-alanine and L-2-aminobutyrate as amino donor and to glyoxylate, pyruvate and 2-oxobutyrate as amino acceptor. The enzyme was identified with rat liver alanine-glyoxylate aminotransferase isoenzyme 2 but not with rat liver alanine-glyoxylate aminotransferase isoenzyme 1 from Ouchterlony double diffusion analysis. Absorption spectra and some kinetic properties of the enzyme were clarified.

Electron spin resonance studies of erythrocytes from spontaneously hypertensive rats and humans with essential hypertension.

The purpose of the present study was to investigate erythrocyte membrane abnormalities in hypertension by means of an electron spin resonance and spin-label technique. The erythrocytes from spontaneously hypertensive rats (SHR) and humans with untreated essential hypertension were examined and compared with their normotensive counterparts, and electron spin resonance spectra were obtained for a fatty spin-label agent (5-nitroxy stearate) incorporated into the erythrocyte membranes. The value of outer hyperfine splitting (2T' parallel) was significantly higher in erythrocytes of SHR and humans with essential hypertension than in erythrocytes of normotensive controls (at 37 degrees C: SHR, 56.14 +/- 0.51 gauss [G], n = 8; Wistar-Kyoto rats, 52.22 +/- 0.86 G, n = 4, p less than 0.01; humans with essential hypertension, 56.94 +/- 0.27 G, n = 11; normotensive subjects, 55.44 +/- 0.36 G, n = 8, p less than 0.01). The order parameter (S) was also increased in the hypertensive rats and humans compared to their respective normotensive controls. When calcium was loaded to erythrocytes with calcium ionophore A23187 (0.9 microM) and CaCl2 (1.0 mM), the parameters of the spectra were increased. These changes were more prominent in the hypertensive groups than in the normotensive controls. These results revealed that the erythrocyte membranes of the hypertensive subjects tolerated different spin motions than those of the normotensive controls in the electron spin resonance study and that membrane fluidity might be decreased in hypertension. Additionally, calcium loading to erythrocytes caused the reduction of membrane fluidity. Therefore, it is suggested that an abnormality of calcium handling at the cellular level might affect physical properties of the biomembranes in hypertension.

Molecular cloning and characterization of a steroid receptor-binding regulator of G-protein signaling protein cDNA.

Steroid hormone receptors are composed of six major functional domains, i.e. the A/B domains as the activation function 1 domain (AF-1), domain C as the DNA-binding domain, domain D as a hinge domain and domain E/F as the ligand-dependent transcriptional domain (AF-2). They regulate gene transcription through interactions with various nuclear factors of their domains, such as AF-1 and AF-2. We have insufficient knowledge of the function of the DNA-binding domain (domain C) except for its DNA-binding function or the hinge domain (domain D). Therefore, we attempted to identify factors interacting with the domains by using a yeast two-hybrid system. Domains C and D of estrogen receptor alpha were used as a bait to isolate cDNA clones from a rat ovary cDNA library. We isolated the cDNA clone of a novel steroid receptor-binding protein bearing the regulator of G-protein signaling (RGS) designated as SRB-RGS. The protein repressed the transcriptional activity of estrogen receptor alpha, suggesting cross-talk of steroid hormones and peptide hormones (or growth factors) for signal transductions mediated by SRB-RGS.

Effects of calcium antagonists on membrane fluidity in hypertension--an electron spin resonance study.

We examined alterations in membrane fluidity of hypertension and the effects of calcium antagonists on these changes by means of an electron spin resonance (ESR) and spin-label technique. Washed erythrocytes from spontaneously hypertensive rats (SHRs; aged 4 and 10-13 weeks) and from patients with essential hypertension, or cultured vascular smooth muscle cells from SHRs were examined and compared with those from normotensive controls. Electron spin resonance spectra for a fatty acid spin label-agent (5-nitroxy stearate) in the membranes were obtained. The values of hyperfine splitting and other parameters of the spectra were significantly higher in erythrocytes from hypertensive rats than in normotensive controls. Similar results were obtained in cultured vascular smooth muscles. These findings indicate that the membrane fluidity might be decreased in hypertension. When calcium was loaded to erythrocytes with Ca-ionophore A23187, the fluidity was decreased. The alternative degrees were greater in hypertensive rats than in controls, and these hypertensive changes were antagonized by diltiazem or verapamil. The results revealed that abnormalities of membrane fluidity in hypertension might be more prominent in the presence of calcium. Further, it is suggested that calcium antagonists could correct these membrane abnormalities in hypertension.

Effects of carbamazepine and valproic acid on brain immunoreactive somatostatin and gamma-aminobutyric acid in amygdaloid-kindled rats.

Somatostatin and gamma-aminobutyric acid (GABA) concentrations were evaluated in the brain of kindled rats treated chronically with carbamazepine and valproic acid. Kindled seizures were almost completely blocked by treatment with carbamazepine, whereas the effect of valproic acid was partial, suppressing only generalized seizures. The duration of after-discharge in amygdala was suppressed by carbamazepine not by valproic acid. Carbamazepine induced a decrease in immunoreactive somatostatin concentration and an increase in GABA concentration in the temporal cortex of kindled rats. Valproic acid induced only an increase in GABA concentration. The results suggest that somatostatin may be associated with the suppression of focal seizure in amygdala and GABA may have a role in the suppression of generalized seizures.

Peroxisome localized human hepatic alanine-glyoxylate aminotransferase and its application to clinical diagnosis.

The subcellular localization of alanine-glyoxylate aminotransferase (EC 2.6.1.44 L-Alanine: glyoxylate aminotransferase) of adult human liver was examined by sucrose density gradient centrifugation. The enzyme sedimented at the same density as catalase, indicating that it was localized in the peroxisomes. Alanine-glyoxylate aminotransferase activity in the liver of patients with cirrhosis was about 65% of that of normal liver or 71% of that from patients with chronic hepatitis, but its activity in the serum of patients with cirrhosis was higher than that from patients with chronic hepatitis. Patterns of activity of alanine-glyoxylate aminotransferase in liver and serum differed from those of aspartate-2-oxoglutarate aminotransferase and ornithine carbamoyltransferase that have a different intracellular location. Serum immunoreactive alanine-glyoxylate aminotransferase (Im-AGT) was measured by enzyme-linked immunoadsorbent assay (ELISA). The Im-AGT levels (mean +/- SEM) in acute (80 +/- 13 micrograms/L) and chronic (72 +/- 4 micrograms/L) hepatitis were higher than those of normal controls (44 +/- 1 micrograms/L). However, the difference between acute and chronic hepatitis was not statistically significant. The level in liver cirrhosis (54 +/- 3 micrograms/L) was lower than those of the hepatitides but higher than that of normal controls. The apparent half-life of serum Im-AGT of patients who underwent liver lobectomy by a microwave tissue coagulation method was approximately 3-4 days.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

Effects of anticonvulsants and gamma-aminobutyric acid (GABA)-mimetic drugs on immunoreactive somatostatin and GABA contents in the rat brain.

Immunoreactive somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) contents in the rat brain were investigated to study chronic effects of the treatment with anticonvulsants, carbamazepine (CBZ), valproic acid (VPA) and phenytoin (PHT). Decreased IR-SRIF levels were found in several brain regions after chronic treatment with VPA and CBZ. GABA concentrations were found to be increased significantly in chronic CBZ and VPA treatment in the rat brain, especially in limbic structures. PHT had no effect on both IR-SRIF and GABA contents in the rat brain. Effects of several GABA-mimetic drugs also were studied on IR-SRIF contents in the rat brain. Aminooxyacetic acid an inhibitor of GABA transaminase, induced a decrease in IR-SRIF concentration in the pyriform and entorhinal cortex, whereas ethanolamine-o-sulfate, another GABA-transaminase inhibitor and muscimol, a GABA receptor agonist had no effect on brain IR-SRIF after acute administration. The present results suggest that endogenous somatostatin has an important role for anticonvulsant properties of CBZ and VPA, but not of PHT. The relationship between the changes in IR-SRIF and the GABA transmitter system in the anticonvulsant action of CBZ and VPA remains to be clarified.

The role of vasopressin, somatostatin and GABA in febrile convulsion in rat pups.

In order to further elucidate a possible role of neuropeptides and GABA in the pathogenesis of febrile convulsions, we studied changes of immunoreactive-arginine vasopressin (IR-AVP), IR-somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) in the rat brain after febrile convulsions induced by ultra-red light (UR). Male Wistar rats at 16 days of age irradiated with UR developed generalized convulsions after 4.9 +/- 0.5 min irradiation. Six rats were killed by microwave irradiation 3 min after UR irradiation prior to convulsion development, and 29 rats were killed either 0 min, 2 h, 6 h, 24 h or 48 h after febrile convulsions. Non-irradiated rats served as controls. The rat brain was dissected into 4 regions; amygdala, hypothalamus, cortex and hippocampus, and subjected to radioimmunoassays. IR-AVP levels in hypothalamus were increased 3 min after UR and decreased at 2 h and 6 h after the convulsions. IR-SRIF levels were increased in cortex and hippocampus at 3 min after UR and 0 min after the convulsions. The GABA content increased in all regions tested at 2 h and 6 h after the convulsions. These results suggest that AVP, SRIF and GABA may be involved in the pathogenesis of febrile convulsions in different ways.

Metabolic inter-organ relations by exercise of fed rat: carbohydrates, ketone body, and nitrogen compounds in splanchnic vessels.

Fed rats were exercised until exhaustion by almost 65% VO2max on a treadmill. In 2.5 min after the exercise, blood was collected from various vessels of the splanchnic bed. Metabolites, glucose, lactate, ketone body, and nitrogencompounds in the plasma, were measured. Glucose excretion from the liver was increased by exercise, but was not significant. The absorption by the kidney decreased to 30% by exercise. Lactate was highly absorbed by the kidney, lower limbs, and digestive tract by exercise. Exercise caused a 200-300% increase of the plasma beta-hydroxybutyrate, but the absorption by the kidney and the lower limbs was decreased. These data suggest that glucose is a good carbon source for the recovery, and that lactate is more useful than glucose, but ketone body is less effective at a very early recovery phase under fed condition. Amino acid balances in each organ except digestive tract were positive showing anabolic conditions of these organs even after exhaustive exercise at fed condition. Most amino acid concentrations in the plasma tended to decrease to 60-90% by exercise. Amino acids were excreted from the digestive tract, and were eventually absorbed by the liver in both rested and exercised rat. The digestive tract, therefore, seems to be a primary amino acids pool to supply them to the liver during the inter meal. Urea excretion from the liver was more than the absorbed ammonia showing that active deamination from amino acids was carrying on. The resulted carbon skeletons of the amino acids might be used for the gluconeogenesis in the liver.

Liver specific kynurenine(alanine):glyoxylate aminotransferase was expressed in kidney cell line.

Kynurenine (Alanine); glyoxylate aminotransferase (AGT) expression plasmid in the COS-7 was constructed. pGV-C was used as a expression vector which contains SV-40 promoter and enhancer. pGV-C and human AGT clone, H1-2, were digested by Hind III/Sma I separately. The AGT fragment was inserted into the digested pGV-C large fragment, The constructed plasmid was named as pGV-AGT. The constructed plasmid was transfected to COS-7 cultured cell by electroporation. The best electroporation condition was checked.

Kinetic properties of human liver tryptophan pyrrolase.

Human liver tryptophan pyrrolase (TPO) activity exhibited substrate level regulation. TPO showed biphasic activity to tryptophan when low ascorbate was used as an activator. The high affinity form (Km for tryptophan: 0.05 mM) was promoted by low ascorbate and low tryptophan. The low affinity form (Km for tryptophan: 0.4 mM) was induced by high concentrations of tryptophan or ascorbate. Both high and low affinity forms showed the same affinity to oxygen. The high affinity form was also induced by pyrroloquinoline quinone, but this effect was decreased by catalase, suggesting the participation of H2O2.

Effect of chronic administration of riboflavin 2',3',4',5'-tetrabutyrate on the hepatic enzymes of fatty acid oxidation in the rat.

Effects of long-term administration of riboflavin, sodium butyrate or riboflavin 2',3',4',5'- tetrabutyrate ( RTB ) on the activities of renal and hepatic enzymes that catalyze the beta-oxidation of fatty acid were determined in the rat. Feeding of riboflavin or sodium butyrate for 5 weeks had no effect on all the enzymes examined. By contrast, feeding of RTB resulted in an increase in the hepatic activity of 3-ketoacyl-CoA thiolase [EC 2.3.1.16] by 50% of the control level, while the activities of renal 3-ketoacyl-CoA thiolase and of hepatic and renal acyl-CoA synthetase [EC 6.2.1.3] and acyl-CoA dehydrogenase [EC 1.3.99.3] remained unaffected. The increase in hepatic 3-ketoacyl-CoA thiolase activity suggests that prolonged RTB administration results in an increased beta-oxidation of fatty acid in the liver, which may explain the reported reduction in the concentration of tryglyceride in plasma during RTB treatment.

[Missense mutations of the butyrylcholinesterase gene in six Japanese patients with low cholinesterasemia: genetic analysis using sera stored in a freezer].

Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.

Large complex formation of the inhibitor of caspase-activated DNase.

Inhibitor of caspase-activated DNase (ICAD) is required for correctly folding of CAD and inhibits nuclease activity of CAD in non-apoptotic cells. From proteomic analysis of the ICAD binding proteins, we revealed that over-expressed flag-ICAD bound other ICAD molecules. Purified recombinant ICAD protein showed three bands, 66 KDa, 132 KDa and 450 KDa, by native-PAGE. ICAD fused with glutathione-S-transferase (GST) was immunoprecipitated with anti-flag antibody from Jurkat cell lysates cotransfected with ICAD fused with either GST or flag expression vectors. When purified recombinant ICAD protein was separated by gel chromatography, the molecular weight of ICAD was detected at approximately 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at approximately 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of approximately 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii.

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.

Fructose 2,6-bisphosphate in rat skeletal muscle during contraction.

Fructose 2,6-bisphosphate and several glycolytic intermediates were measured in two rat muscles, extensor digitorum longus and gastrocnemius, which were electrically stimulated in situ. Both the duration and the frequency of stimulation were varied to obtain different rates of glycolysis. There was no relationship between fructose 2,6-bisphosphate content and the increase in tissue lactate in contracting muscle. However, in gastrocnemius stimulated at low frequencies (less than or equal to 5 Hz), there was a 2-fold increase in fructose 2,6-bisphosphate at 10s, followed by a return to basal values, whereas lactate increased only after 1 min of contraction. The concentrations of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates were all increased during the 3 min stimulation. During tetanus (frequencies greater than or equal to 10 Hz) fructose 2,6-bisphosphate was not increased, whereas glycolysis was maximally stimulated and resulted in an accumulation of tissue lactate, mostly from glycogen. The concentrations of hexose 6-phosphate increased continuously during the 1 min tetanus, whereas fructose 1,6-bisphosphate was increased at 10s and then decreased progressively. It therefore appears that fructose 2,6-bisphosphate does not play a role in the stimulation of glycolysis during tetanus; it may, however, be involved in the control of glycolysis when the muscles are stimulated at low frequencies for short periods of time.

Purification, characterization and identification of tryptophan aminotransferase from rat brain.

Tryptophan aminotransferase was purified from rat brain extracts. The purified enzyme had an isoelectric point at pH 6.2 and a pH optimum near 8.0. On electrophoresis the enzyme migrated to the anode. The enzyme was active with oxaloacetate or 2-oxoglutarate as amino acceptor but not with pyruvate, and utilized various L-amino acids as amino donors. With 2-oxoglutarate, the order of effectiveness of the L-amino acids was aspartate >> 5-hydroxytryptophan > tryptophan > tyrosine > phenylalanine. Aminotransferase activity of the enzyme towards tryptophan was inhibited by L-glutamate. Sucrose density gradient centrifugation gave a molecular weight of approx. 55,000. The enzyme was present in both the cytosol and synaptosomal cytosol, but not in the mitochondria. The isoelectric focusing profile of tryptophan: oxaloacetate aminotransferase activity was identical with that of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) activity, with both subcellular fractions. On the basis of these data, it is suggested that the enzyme is identical with the cytosol aspartate: 2-oxoglutarate aminotransferase.