Developmental regulation of D-beta-hydroxybutyrate dehydrogenase in rat liver and brain

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-beta-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

Prostaglandin dehydrogenase activity of purified rat liver 3 alpha-hydroxysteroid dehydrogenase

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol displays 9, 11, and 15-hydroxyprostaglandin dehydrogenase activity. Using [14C]-PGF2 alpha as substrate the products of this reaction were separated by TLC and identified by autoradiography as PGE2 and PGB2. The purified enzyme catalyzes this reaction at a rate 200 times faster than cytosol. This corresponds to the rate enhancement observed when the enzyme is purified from cytosol using androsterone (a 3 alpha-hydroxysteroid) as substrate and suggests that it may represent a major 9-hydroxyprostaglandin dehydrogenase in this tissue. Although the 3 alpha-HSD has many properties in common with the 9-hydroxyprostaglandin dehydrogenase of rat kidney, rat kidney contains no protein that is immunodetectable with polyclonal antibody raised against the purified 3 alpha-HSD.     

Cyclic succinic dehydrogenase activity in rat liver mitochondria

Succinic dehydrogenase activity was assayed in isolated rat liver mitochondria. Rats had been exposed for at least two weeks to a 24 hour feeding-fasting schedule, 5 hours feeding, 19 hours fasting. Enzyme activity was determined at nine specific hours over a 24 hour period. Lowest enzyme activity occured six hours after the feeding cages had been closed. The highest activity, which was 158 percent greater than the lowest activity, occurred during the next feeding period. It is concluded that time of sacrifice is an important consideration when determining succinic dehydrogenase activity.     

N-acetyl-beta-D-glucosaminidase activity in serum, kidney and liver of streptozotocin-induced diabetic rat

Serum N-acetyl-beta-D-glucosaminidase (NAG) activity in streptozotocin-induced diabetic rats was significantly increased. There was neither a difference in total NAG activity in kidney and liver, nor in optimal pH of NAG in serum, kidney and liver between diabetic and control rats. The ratio of the thermounstable fraction of NAG increased in diabetic kidney and liver, while there was no difference in thermostability of between diabetic and control rats. Isoelectricfocusing of diabetic serum NAG indicated an increase in the neutral form. That of kidney and liver NAG indicated an increase in the acid form. These results may suggest that NAG clearance from the serum is decreased diabetic state.     

Effect of caffeine on ornithine metabolism in rat brain, liver and kidney

Prolonged treatment with caffeine promotes in rats an increase of liver ornithine carbamyltransferase activity (14-day treatment). In contrast, arginase activity is already reduced in brain and kidney after 10 days, and in the liver much later (17 days). Ornithine transaminase activity was increased in both liver and kidney, while in the brain it was reduced (17 days). Ornithine decarboxylase activity showed only minor modifications in kidney, while it was unchanged in brain. Of the polyamines, only spermidine was significantly modified, being increased in brain, decreased in liver and kidney. Although these results do not explain the mechanism of the modification of brain arginine and ornithine concentration promoted by caffeine, they point to further marked effects, i.e. on OAT activity and on spermidine concentration, which could have a relevant metabolic role.     

Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle

Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Summary Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.Supported by grants of the Forschungsförderung des Landes Nordrhein-Westfalen, No. 40002585 and the Verein der Freunde und Förderer der Universität KölnDedicated to Professor Dr. Dietrich Eichner on the occasion of his 65th birthday     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

Comparison of D-beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria

The properties of D-beta-hydroxybutyrate dehydrogenase (BDH) from rat liver and brain mitochondria were compared to determine if isozymes of this enzyme exist in these tissues. The BDHs from these tissues behaved similarly during the purification process. The enzymes were indistinguishable by sodium dodecyl sulfate-polyacrylamide or acid-urea-polyacrylamide gel electrophoresis and they had identical isoelectric points. The BDHs from rat liver and brain were also quite similar in functional parameters determined by kinetic analysis and phospholipid activation of apo-BDH (i.e., the lipid-free enzyme). Antiserum against rat liver BDH inhibited both enzymes to an equivalent extent in a titration assay. The enzymes had similar patterns of peptide mapping by partial digestion with Staphylococcus aureus V8 protease, followed by immunoblotting using antiserum against the liver enzyme. These results suggest that the BDHs in rat liver and brain are very similar and possibly identical.