Conversion of thyroxine into tri-iodothyronine by rat liver homogenate.

By using a highly specific radioimmunoassay the formation of tri-iodothyronine by the deiodination of thyroxine was studied in rat liver homogenate. Several observations suggest that the reaction observed is enzymic in nature. Pre-heating the homogenate for 30 min at 56 degrees C completely abolished conversion of thyroxine into tri-iodothyronine; the component of rat liver homogenate responsible could be saturated with substrate; iodotyrosines displayed competitive activity. Between 0 degrees and 37 degrees C, the tri-iodothyronine-production rate was positively correlated with incubation temperature. The addition of NAD+ enhanced conversion into tri-iodothyronine, which suggests that an oxidative mechanism is involved. 5-Propyl-2-thiouracil and 6-propyl-2-thiouracil, both known to prevent deiodination in vivo, greatly decreased the deiodiantion activity of rat liver homogenate.     

Phosphatidate phosphohydrolase and palmitoyl-coenzyme A hydrolase in cardiac subcellular fractions of hyperthyroid rabbits and cardiomyopathic hamsters

Activities of phosphatidate phosphohydrolase and palmitoyl-CoA hydrolase were determined in cardiac subcellular fractions prepared from rabbits which has received tri-iodothyronine and from hamsters with hereditary cardiomyopathy (strain BIO 14.6). 1. Both mitochondrial and microsomal fractions of hyperthyroid rabbit hearts produced 4-5 times as much diacylglycerol 3-phosphate from glycerol 3-phosphate and palmitate as did those of euthyroid hearts. 2. Phosphatidate phosphohydrolase, measured with phosphatidate emulsion, was activated by 1mm-Mg(2+) in all but the mitochondrial fraction of euthyroid rabbit hearts. The activation was more pronounced in subcellular fractions isolated from hyperthyroid hearts, so that the measured activities were significantly increased above those of the controls. The highest activity was found in the microsomal and lysosomal fractions. 3. In the absence of Mg(2+) during incubation, the difference in phosphohydrolase activities between eu- and hyper-thyroid states was not significant. 4. The phosphohydrolase of subcellular fractions of control hamsters did not respond to addition of 0.5-8.0mm-Mg(2+). The enzyme from cardiomyopathic hearts was slightly inhibited by this bivalent cation and therefore significant increases in activity were observed only in the absence of Mg(2+) from the assay system. 5. The rate of reaction by soluble phosphatidate phosphohydrolase was similar regardless of the nature of the substrate. Both when microsomal-bound phosphatidate was used as the substrate and when phosphatidate suspension was used, the activity of soluble enzyme was lower than that of the microsomal and lysosomal enzymes measured with phosphatidate suspension; this was especially so when the assay was carried out in the absence of Mg(2+). Neither tri-iodothyronine nor cardiomyopathy influenced the soluble phosphohydrolase activity in the two species. 6. Neither tri-iodothyronine nor cardiomyopathy significantly changed palmitoyl-CoA hydrolase activities in subcellular fractions. 7. Microsomal diacylglycerol acyltransferase and myocardial triacylglycerol content were also unchanged in the hyperthyroid state.     

Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.     

Early stimulation of rat liver microsomal protein synthesis after tri-iodothyronine injection in vivo.

In an effort to determine the physiological significance of previous studies showing stimulation of microsomal protein synthesis by thyroxine added in vitro, an early effect of tri-iodothyronine injected in vivo was sought. Tri-iodothyronine (25 micrograms/100 g) administered to euthyroid rats stimulated microsomal protein synthesis in vitro within 3--6 h. This effect occurred much earlier than the 26 h lag previously reported after tri-iodothyronine administration to hypothyroid rats. This early effect of tri-iodothyronine on protein synthesis is prevented by alpha-amanitin, suggesting that it is dependent on RNA synthesis. The failure to find a direct effect in vivo of tri-iodothyronine on translation casts doubt on the physiological significance of previous studies that have shown a direct stimulation of translation by thyroxine added in vitro.     

Re-examination of the subcellular localization of thyroxine 5''-deiodination in rat liver.

We describe the existence of at least two thyroxine 5'-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5'-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5'-deiodination. However, this process is not mediated by only a single enzyme.     

Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver

Sphingosine-1-phosphate lyase is responsible for the ultimate step in sphingolipid breakdown, converting phosphorylated long chain bases into ethanolamine phosphate and a fatty aldehyde. Using tritiated dihydrosphingosine-1-phosphate, prepared enzymatically from [4,5-3H]dihydrosphingosylphosphocholine, we have reinvestigated the subcellular distribution of this enzyme in rat liver. Upon cell fractionation by differential centrifugation, the enzyme showed a microsomal distribution. Further separation of the microsomal fraction by sucrose gradient centrifugation confirmed an association with the endoplasmic reticulum. By means of constrained nonlinear regression, no evidence for a significant association with mitochondrial membranes, as reported previously (Stoffel, W., LeKim, D., and Sticht, G. (1969) Hoppe Seyler's Z. Physiol. Chem. 350, 1233-1241), nor with other cell compartments was found. The lyase activity, which appeared to be sensitive to different detergents, but not to Triton X-100, was not latent. It could be solubilized with Triton X-100, but not by high ionic strength, indicating that it is an integral membrane protein whose catalytic site is most probably exposed to the cytosol. Treatment of intact microsomal vesicles with trypsin or thermolysin inactivated the lyase activity, confirming that its catalytic site(s) or other domains essential for activity face the cytosol.     

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.     

Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.     

The control of CTP:choline-phosphate cytidylyltransferase activity in pea (Pisum sativum L.).

Several possible control mechanisms for CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity in pea (Pisum sativum L.) stems were investigated. Indol-3-ylacetic acid (IAA) treatment of the pea stems decreased total cytidylyltransferase activity but did not affect its subcellular distribution. Oleate (2 mM) caused some stimulation of enzyme activity by release of activity from the microsomal fraction into the cytosol, but neither phosphatidylglycerol nor monoacyl phosphatidylethanolamine had an effect on activity or subcellular distribution. A decrease in soluble cytidylyltransferase protein concentrations was found in IAA-treated pea stems, but this was not sufficient to account for all of the decrease in cytidylyltransferase activity. A 50% inhibition of enzyme activity could be obtained with 0.2 mM-CMP, which indicated possible allosteric regulation. Similar inhibition was obtained with 1.5 mM-ATP, but other nucleotides had no effect. The cytidylyltransferase enzyme protein was not directly phosphorylated, and the inhibition with 1.5 mM-ATP occurred with the purified enzyme, thus excluding an obligatory mediation via a modulator protein. The results indicate that the cytosolic form of cytidylyltransferase is the most important in pea stem tissue and that the decrease in cytidylyltransferase activity in IAA-treated material appears to be brought about by several methods.     

On the distribution of aldolase isoenzymes in subcellular fractions from rat brain

As an extension of previous studies on the adsorption of aldolase (EC 4.1.2.13) in nervous tissue, the main features of the subcellular localization of this enzyme in rat brain have been investigated. The major portion of the aldolase activity in homogenates of this tissue was demonstrated to be present in association with the particulate material, and a differential distribution of the AC isoenzymes was evident between the membranes and the cytosol. Some of the enzyme which was associated with the particulate fraction was shown to be occluded rather than absorbed to the membranes. This type of association was evident in the nuclear and mitochondrial fractions, in particular, with the occluded enzyme presenting an isoenzyme content high in C-type activity, and similar to that of the cytosol. The microsomal fraction contained a high proportion of enzyme in the bound form. Isoenzyme analysis of the enzyme in this microsomal fraction revealed a preferential association between the particulate material and A-type aldolase activity. A purified membrane fraction was prepared from the primary microsomal fraction, and identified as the main site of aldolase binding. The significance of the differential binding of aldolase isoenzymes and its localization amongst the subcellular fractions of rat brain have been discussed in relation to the structural and metabolic features of this tissue, and the coupling of energy producing sequences with energy requiring processes.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Membrane-associated pyruvate kinase in developing guinea-pig liver.

During analysis of pyruvate kinase distribution in developing guinea-pig liver it was observed that a substantial proportion of the activity remained associated with the microsomal membrane fraction ('microsomes'). Although some of this could be removed by washing with sucrose, the majority required detergent treatment for liberation, and even then at least one-half remained attached to the microsomes. Estimates of the contribution of this fraction to total cell pyruvate kinase activity indicated that it was more than 50% of the total, and this is likely to be an underestimate because of the continued latency of the enzyme even in the presence of detergent. The susceptibility of the microsomal enzyme, whether released by detergent or sucrose washing, to inactivation by Triton X-100 suggested it to be different from the cytosolic enzyme, which was stable under such conditions. (The microsomal enzyme required the presence of additional protein, such as bovine serum albumin, to maintain stability.) This view was confirmed by DEAE-cellulose chromatography and particularly isoelectric focusing, where the microsomal enzyme was shown to consist of at least four forms, which were distinctly different from those in the cytosol. Those data and the kinetic properties of the four forms in the membrane fraction indicate that the microsomal pyruvate kinase could consist of four counterparts to the cytosolic isoenzyme forms. These results are discussed in relation to the two possible explanations for the phenomenon (not mutually exclusive): that the more hydrophobic membrane forms are precursors of the cytosolic enzyme and that they may be part of functional glycolytic pathway in the microsomes of developing liver.     

Purification and properties of prostaglandin D synthetase from rat brain.

The prostaglandin D synthetase system was isolated from rat brain. Prostaglandin endoperoxide synthetase solubilized from a microsomal fraction catalyzed the conversion of arachidonic acid to prostaglandin H2 in the presence of heme and tryptophan. Prostaglandin D synthetase (prostaglandin endoperoxidase-D isomerase) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2 was found predominantly in a cytosol fraction and was purified to apparent homogeneity with a specific activity of 1.7 mumol/min/mg of protein at 24 degrees C. The enzyme also acted upon prostaglandin G2 and produced a compound presumed to be 15-hydroperoxy-prostaglandin D2. Glutathione was not required for the enzyme reaction, but the enzyme was stabilized by thiol compounds including glutathione. The enzyme was inhibited by p-chloromercuribenzoic acid in a reversible manner. The purified enzyme was essentially free of the glutathione S-transferase activity which was found in the cytosol of brain.     

Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro.

The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.     

Prostaglandins in human breast cancer. Identification of a cytosolic prostaglandin-9-keto-reductase activity

Endogenous sources of prostaglandin production in human breast tumors were investigated by radioimmunoassay analysis of PGE2 and PGF2a productions and 3H-PGE2 conversion. PG synthetase located within the microsomal fraction mainly produced PGE2, while little PGF2a synthesis occured. In cytosol preparations. PGE2 is converted into PGF2a. In 15 tumor specimens, no relationship was observed between PGE2 production and the metabolic activity which varied widely from sample to sample. These findings demonstrate the presence of PG-9-keto-reductase in the cytosol from human breast tumors. A way of PGE2 inactivation by this enzyme is suggested since no less polar PGE2 metabolites were detected. It is concluded that PGE2 production by the microsomes will reflect the PG synthetase activity of a given human mammary carcinoma while metabolic conversion of PGE2 within the cytosol reflects the metabolic activity of the same sample. Both activities were otherwise apparently unlinked.     

The subcellular distribution of beta-carotene in bovine corpus luteum

The distribution of beta-carotene was determined in various subcellular fractions of bovine corpus luteum. It was found in significant amounts in all subcellular fractions examined including nuclear, mitochondrial, microsomal, cytosolic, and floating lipid. Much of the beta-carotene found in the crude nuclear and mitochondrial fractions was loosely bound and could be removed with repeated washings. In contrast, the microsomal beta-carotene could only be removed by detergent extraction suggesting that it is an integral component of this membrane preparation. In the cytosol fraction beta-carotene was bound to high-molecular-weight protein(s), quite possibly a plasma-derived lipoprotein. The subcellular distribution of beta-carotene in corpus luteum is quite similar to the distribution of its metabolite, retinol, in liver. This finding coupled with other recently published data suggests that beta-carotene could play a distinct role in corpora lutea function.     

Solubilization and partial characterization of rat epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase).

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.     

Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Distribution and properties of aldehyde dehydrogenase in regions of rat brain

Abstract: NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3) were isolated from various subcellular organelles as well as from different regions of rat brain. The mitochondrial, microsomal, and cytosolic fractions were found to contain 40%, 28%, and 12%, respectively, of the total aldehyde dehydrogenase (5.28 ± 0.44 nmol NADH/min/g tissue) found in rat brain homogenate when assayed with 70 μ. M propionaldehyde at pH 7.5. The total activity increased to 17.3 ± 2.7 nmol NADH/min/g tissue when assayed with 5 m M propionaldehyde. Under these conditions the three organelles contained 49%, 23%, and 9%, respectively, of the activity. The enzyme isolated from cytosol possessed the lowest K _m. The molecular weight of the enzyme isolated from all three subcellular organelles was ∼100,000. Four activity bands were found by electrophoresis of crude homogenates, isolated mitochondria, or microsomes on cellulose acetate strips. Cytosol possessed just two of the forms. The total activity was essentially the same in homogenates obtained from cortex, subcortex, pons-medulla, or cerebellum. Further, the enzyme had the same molecular distribution and total activity in each of these four brain regions. Disulfiram was found to be an in vivo and in vitro inhibitor of the enzymes obtained from these brain regions. Mercaptoethanol, required for the stability of the enzyme, reversed the inhibition produced by disulfiram. The effect was greater for enzyme isolated from cytosol than from mitochondria. Calculations led to the prediction that aldehydes such as acetaldehyde are oxidized in cytosol.