Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain

In order to further understand the role of enzymes degrading Thyrotropin Releasing Hormone (TRH, pglu-his-proNH₂) and metabolites, we studied their subcellular distribution in rat brain. Brain tissue was homogenized in 0.32 M sucrose, tris-HCl 0.01 M pH 7.4 and fractionated by differential and discontinuous gradient centrifugation; [³H]pro-TRH was incubated with the various subcellular fractions and the extent of degradation of each metabolite was measured after separation by thin layer chromatography. Several markers were simultaneously measured (lactate dehydrogenase, 5′-nucleotidase and hexosaminidase) to determine the pattern of distribution of the subcellular organelles. The post-proline cleaving enzyme responsible for pglu-his-pro formation and pyroglutamate amino-peptidase (which requires sulphydryl compounds for maximal activity) were found in cytosol but were barely detectable in the soluble component of synaptosomes; pyroglutamate aminopeptidase (dependent on metals) and post-proline dipeptidyl amino peptidase were found on the membranes of synaptosomes; imido peptidase was not enriched in any particular fraction.These data are consistent with the hypothesis that membrane-bound pyroglutamate aminopeptidase is responsible for TRH degradation once released into the synaptic cleft and that the post-proline dipeptidylaminopeptidase may participate in the extracellular catabolism of his-proNH₂ before it cyclizes to his-pro-DKP. They also suggest that post-proline cleaving enzyme and soluble pyroglutamate aminopeptidase may not play an important role in the regulation of TRH levels in nerve endings.     

Subcellular distribution of adenosine system enzymes in the hypothalamus and hippocampus of the rat brain

The subcellular distribution of 5'-nucleotidase and adenosine deaminase in rat brain hypothalamus and hippocampus was studied. In the hippocampus the 5'-nucleotidase activity was shown to be much higher than in the hypothalamus, while the adenosine deaminase activity, contrariwise, is nearly two times as high as that in the hypothalamus. During the analysis of subcellular distribution 5'-nucleotidase and adenosine deaminase were detected in all fractions under study, i. e., in nuclear, soluble, myelin fractions as well as in synaptic membranes, synaptosomes and "pure" mitochondria. The highest 5'-nucleotidase activity was found in the myelinic and synaptic fractions both in the hypothalamus and in the hippocampus. The highest adenosine deaminase activity was detected in the soluble fraction of the above structures. The enzyme activity in synaptic membranes and synaptosomes was nearly two times as low.     

Subcellular distribution of lysophosphatidylinositol and lysophosphatidylcholine acyltransferases in rat brain.

Lysophosphatidylinositol acyltransferase utilizing arachidonoyl CoA and lysophosphatidylcholine acyltransferase utilizing linoleoyl CoA were measured in subcellular fractions of rat brain. In general, the distribution of activities paralleled that of NADPH--cytochrome c reductase. It is concluded that the endoplasmic reticulum is the major site of these acyltransferase activities in rat brain.     

Subcellular distribution of marker enzymes and of neurotoxic esterase in adult hen brain.

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P₃) which was also enriched in 5′-nucleotidase (5′-ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P₃ fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′-nucleotidase and glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P₂ fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.     

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain.

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.     

Subcellular localization of enzymes oxidizing citrate in the rat brain

¹RSA was calculated as the ratio: percentage of total recovered activity found in fraction to percentage of total protein found in the same fraction.     

Subcellular distribution of diacylglycerol lipase in rat and mouse brain

Rat Brain has a lipase which hydrolyzes diacylglycerol at an optimal pH of 4.8 (1). The subcellular distribution of this acid diacylglycerol lipase was studied in brain tissue of rats and mice; in the latter case neurological mutants and their normal controls were used. Several other acidic hydrolases were employed as normal controls were used. Several other acidic hydrolases were employed as lysosomal markers. In mouse brain, the specific activity which is about 50-100 times lower than in rat brain, was greatest in the lysosomal fraction. In contrast, no enrichment of DG-lipase was observed in any subcellular fraction of the active enzyme of rat brain. Activities were about equally distributed in the microsomal, myelin-synaptosomal and lysosomal fractions.     

Subcellular distribution of hepatic bile acid-conjugating enzymes.

1. The subcellular location of enzymes conjugating bile acids with glycine or taurine was investigated by centrifugation of rat liver homogenates. 2. [14C]Cholic acid-conjugating activity was predominantly associated with the soluble-microsomal region of the gradient after centrifugation in a Ti-15 zonal rotor but the bulk of the conjugating activity sedimented with mitochondrial-lysosomal fractions in differential pelleting experiments. 3. Cholate: CoA ligase (EC 6.2.1.7) and cholyltransferase (EC 2.3.1) were not enriched in purified Golgi or plasma-membrane fractions. Cholate: CoA ligase was distributed evenly between rough- and smooth-surfaced microsomal subfractions but cholyltransferase showed a dual soluble-rough microsomal activity distribution. 4. Sedimentation of cholyltransferase in mitochondria-enriched fractions prepared by differential centrifugation appears to be an artefact of sedimentation of rough microsomal membranes in mitochondrial fractions. 5. The subcellular distribution of bile acid-conjugating enzymes is discussed with reference to hepatic processing of bile acids.     

Subcellular distribution of taurine and cysteinesulphinate decarboxylase in developing rat brain

The concentration of taurine and the activities of cysteinesulphinate decarboxylase and glutamate decarboxylase have been measured in rat brain. During development, taurine exhibited a decrease in concentration unrelated to the activity of cysteinesulphinate decarboxylase which increased during the same period. The distribution of taurine in subcellular fractions of adult and 7-day-old rat brain was typical of most amino acids, whereas half of the cysteinesulphinate decarboxylase activity was found in the nerve-ending cytoplasm. In anatomical distribution, taurine displayed great regional heterogeneity but both cysteinesulphinate decarboxylase and glutamate decarboxylase were more evenly distributed. Hypertaurinaemia was shown to have no effect on the entry of glycine into the brain or on its utilization in protein synthesis.     

Subcellular distribution of aromatic amino acid transaminases in rat brain

Abstract— The subcellular distributions of tyrosine transaminase, DOPA transaminase, tryptophan transaminase and 5-hydroxytryptophan (5-HTP) transaminase were studied in rat brain.

• 1 For all of these transaminases 60-81 per cent of the total activities were found in the crude mitochondrial fraction. Tyrosine transaminase was the most active enzyme.

• 2 Tyrosine transaminase and DOPA transaminase had very similar distributions in all fractions, but the distribution of tryptophan transaminase and 5-HTP transaminase differed in the microsomal (Mic) and synaptic vesicle (M₂) fractions. Only 5-HTP transaminase was highly concentrated in the M₂ fraction.

• 3 DOPA transaminase was inhibited by dopamine and 5-HT, but these compounds had no effect on 5-HTP transaminase. Both enzymes were completely inhibited by m-hydroxybenzoyloxyamine.

     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.