Aspartate aminotransferase isozymes from rabbit liver. Purification and properties

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.     

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.     

Differentiated pressor and sympathetic responses to dual brain stimulation: ventromedial hypothalamus versus locus coeruleus

Sensitivity of the ventromedial hypothalamus (VMH) to electrical stimulation was compared with that of the locus coeruleus (LC) in urethane-anesthetized rats. Based not only on current strengths required to elicit threshold effects, but also on magnitude of pressor responses to suprathreshold stimulation, the LC was consistently more sensitive than the VMH. Despite this greater pressor sensitivity, splanchnic nerve firing increased almost equally upon stimulation of either brain area. Similar comparisons made in other rats following bilateral adrenalectomy or pretreatment with a vasopressin antagonist showed no significant alteration of pressor and sympathetic responsiveness to stimulation of either the LC or the VMH. When frequency of neural firing was recorded from a lumbar sympathetic trunk instead of the splanchnic nerve, increases in sympathetic nerve activity produced by LC stimulation were significantly larger than those produced from the VMH. The results suggest that greater pressor sensitivity of the LC is due, at least in part, to stronger constriction in vascular beds innervated by the lumbar sympathetic chains.     

Effects of short-term treatment with calcium on the parathyroid gland of the rat,under particular consideration of the alteration of storage granules

Summary Short-term effects of CaCl₂-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl₂, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded (i) that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and (ii) that these processes may be accelerated during hypercalcemia.     

Mechanism of biliary secretion of membranous enzymes: bile acids are important factors for biliary occurrence of gamma-glutamyltransferase and other hydrolases

To elucidate the mechanism of biliary occurrence of gamma-glutamyl transferase [EC 2.3.2.2] and alkaline phosphatase [EC 3.1.3.1], the effect of bile acids on the biliary level of these enzymes was studied in vivo and in vitro. Following intravenous administration of taurocholate, the activities of both enzymes in rat bile increased markedly with a concomitant increase in the excretion of the bile acid. The biliary levels of these enzymes increased to reach a maximum at 10-20 min after administration of the bile acid and decreased thereafter. Right-side-out oriented rat liver canalicular membrane vesicles which localize gamma-glutamyltransferase, aminopeptidase M and alkaline phosphatase on their outer surface (Inoue, M., Kinne, R., Tran, T., Biempica, L., & Arias, I.M. (1983) J. Biol. Chem. 258, 5183-5188) were prepared. Upon incubation of the vesicles with either intact or heat-treated bile samples, the membranous enzymes were released from the vesicles in a time-dependent manner. Incubation of these vesicles with physiological concentrations of taurocholate also solubilized these enzymes from the membranes. Affinity chromatographic analysis on concanavalin A-Sepharose revealed that the transferase thus solubilized retained the hydrophobic domain responsible for anchoring the enzyme to membrane/lipid bilayers. These results indicate that bile acid(s) excreted into the bile canalicular lumen solubilized these enzymes from the apical membrane surface of the biliary tract cells by their detergent action.     

Interaction of digitonin and its analogs with membrane cholesterol

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible sensory receptor of nonadrenergic inhibitory nervous system

To determine the sensory receptor of the nonadrenergic inhibitory nervous system (NAIS), 22 cats were anesthetized and serotonin was continuously administered (50-250 micrograms.kg-1.min-1 iv) to increase pulmonary resistance (RL) to 377 +/- 57% (SE) of the control value. We then 1) mechanically irritated the trachea, 2) intravenously administered capsaicin (5 micrograms/kg), or 3) induced hypoxia (arterial PO2 30-40 Torr) to stimulate irritant and bronchial C-fiber receptors, pulmonary C-fiber receptors, or the carotid body (chemoreceptors), respectively. After treatment with atropine (3 mg/kg iv) and propranolol (2 mg/kg iv), the serotonin-induced change in RL was reduced by 58.6 +/- 14.3% by mechanical irritation and 63.3 +/- 12.1% by intravenous capsaicin. However, hypoxia produced no dilatation of the airways. In further experiments, we employed capsaicin inhalation to stimulate bronchial C-fiber receptors. Inhaled capsaicin (0.1%, for 5 breaths) also reduced RL by 79.2 +/- 9.2% of the elevated value, after atropine and propranolol. Treatment with a ganglionic blocking agent, hexamethonium (2 mg/kg iv), abolished bronchodilator responses, implying that a reflex pathway through vagal nerves is involved in this phenomenon. These results suggest that pulmonary and bronchial C-fiber receptors may be involved as sensory receptors in NAIS reflex bronchodilatation.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Accuracy of endometrial aspiration in the diagnosis of endometrial cancer

A study was performed to evaluate the cytologic criteria for recognizing endometrial cancer and to determine the accuracy of endometrial aspiration in its detection. In addition to the conventional cytologic criteria for the diagnosis of endometrial cancer, the irregular chromatin distribution around the macronucleoli proved to be a useful criterion. Using all criteria, cytology was reported as positive in 18 of 19 patients (94.7%) with endometrial cancer. In 856 of 12,563 high-risk outpatients at Kinki University Hospital, aspiration using the Masubuchi apparatus was carried out in screening for endometrial cancer. Cancer was detected in 18 patients (2.1%), with 94.7% of the cancers detected by the cytologic screening. This result indicates that endometrial aspiration using the Masubuchi apparatus is a reliable and safe method of screening for endometrial cancer.     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)