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Biomedical subjects

E H Harrison

Publications and source records attributed to E H Harrison.

51 records · Page 3Linked to original sources
PubMed1988

Perfluoro-n-decanoic acid: induction of peroxisomal beta-oxidation by a fatty acid with dioxin-like toxicity.

Perfluoro-n-decanoic acid (PFDA) produces toxic effects in rodents similar to those caused by 2,3,7,8-tetrachloro-dibenzo-p-dioxin. A single, intraperitoneal dose (50 mg/kg) of PFDA to Sprague-Dawley rats caused disruption of the endoplasmic reticulum, mitochondrial swelling and increases in intracellular lipid droplets in hepatocytes similar to effects reported previously in dioxin toxicity. PFDA treatment led to large decreases in the activity of plasma membrane alkaline phosphodiesterase and mitochondrial cytochrome c oxidase without affecting lysosomal N-acetyl-beta-glucosaminidase, endoplasmic reticulum NADPH-cytochrome c reductase or peroxisomal catalase activities. PFDA treatment led to moderate peroxisome proliferation and to very large (20-40-fold) increases in the activity of fatty acyl-CoA oxidase, the rate-limiting enzyme in the peroxisomal system of fatty acid beta-oxidation.

Animals↗
PubMed1988

Properties and subcellular localization of myocardial fatty acyl-coenzyme A oxidase.

The properties and subcellular localization of fatty acyl-CoA oxidase (FAO) were studied in rat heart homogenates. After differential centrifugation, FAO was sedimentable and enriched in a "light-mitochondrial" fraction. FAO had a pH optimum of 8-9. Among straight-chain, saturated fatty acyl-CoAs, the enzyme showed a marked preference for medium chain substrates (C12 greater than C10 = C8 greater than C16 = C14 greater than C6) over a concentration range up to 100 microM. No activity was observed with C4-CoA. The apparent Michaelis constant (Km) for C12-CoA was 5-10 microM. After removal of nuclei by low-speed centrifugation, combined subcellular particle preparations were obtained by high-speed centrifugation and layered on linear density gradients of metrizamide. After density equilibration, FAO showed a symmetric distribution centered at p = 1.16-1.18, like that of the enzyme catalase, a marker for microperoxisomes. In contrast, enzyme markers for mitochondria, lysosomes, sarcolemma, and sarcoplasmic reticulum were recovered in low-density regions of the gradient. These results provide a direct demonstration of fatty acyl-CoA oxidase in cardiac tissue and its association with microperoxisomes.

Acyl-CoA Oxidase↗
PubMed1987

Subcellular localization of retinoids, retinoid-binding proteins, and acyl-CoA:retinol acyltransferase in rat liver.

Studies were conducted to define the subcellular localization of endogenous retinoids (vitamin A), retinoid-binding proteins, and acyl-CoA:retinol acyltransferase (ARAT) in liver and to determine whether their distributions were affected by hepatic vitamin A content. Quantitative subcellular fractionation techniques were used. Rats were fed purified diets either containing or lacking vitamin A to obtain animals with total retinoid stores ranging from 0.5 to 172 micrograms of retinol equivalent per gram of liver. Liver homogenates were fractionated by differential centrifugation to yield nuclear (N), mitochondrial-lysosomal (ML), microsomal (P), and high-speed supernatant (S) fractions. N, ML, and P were washed two more times by resuspension and centrifugation to remove constituents bound nonspecifically. S was further resolved into "floating lipid" and underlying "cytosol" by prolonged ultracentrifugation. The distributions of marker constituents were not affected by vitamin A status. Most of the retinyl ester in the liver was recovered in the S fraction where it was entirely (greater than 95%) associated with floating lipid. About half of the total free retinol was also recovered in the S fraction, but it was mostly (2/3) associated with cytosol per se. A substantial portion (30%) of the free retinol was recovered in the 3 X -washed microsomal (P) fraction. Sufficient binding capacity for retinol was present in both P (as retinol-binding protein) and S (as cellular retinol-binding protein) to quantitatively account for the amounts of free retinol present in the two fractions. ARAT activity in the liver was distributed among the subcellular fractions in a manner identical with an endoplasmic reticulum marker enzyme (NADPH-cytochrome C reductase).(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed1985

Local anesthetic properties of opioids and phencyclidines: interaction with the voltage-dependent, batrachotoxin binding site in sodium channels.

[3H]Batrachotoxinin-A 20-alpha-benzoate ([3H]BTX-B) binds specifically and with high affinity (Kd = 30 nM) to a site on voltage-dependent Na+ channels. Compounds with local anesthetic activity inhibit the binding of [3H]BTX-B by a mutually exclusive, allosteric mechanism. The potential local anesthetic potency of a series of 23 opioids and phencyclidine-like compounds has been estimated by their inhibition of [3H]BTX-B binding to Na+ channels in a preparation of synaptoneurosomes from guinea pig cerebral cortex. The potency of these compounds were also tested as inhibitors of the specific binding of [3H]phencyclidine ([3H]PCP) to a high affinity site on rat brain membranes. Opioids such as morphine and codeine show little affinity for the [3H]BTX-B binding site or for the [3H]PCP binding site. Other analgesics, many of the PCP-like compounds and dioxadrol derivatives are potent versus [3H]BTX-B binding and display both stereospecificity and high affinity towards the PCP-binding site. However, there was no correlation between local anesthetic potency assessed as antagonism of [3H]BTX-B binding and affinity towards the PCP site. Five classical local anesthetics had no affinity for the PCP-site, but did displace [3H]BTX-B from its binding site.

Anesthetics, Local↗
PubMed1984

Action of clofibrate and its analogs in rats. Dissociation of hypolipidemic effects and the induction of peroxisomal beta-oxidation.

Experiments were conducted to compare directly the effects of clofibrate (ethyl 2-(p-chlorophenoxy)isobutyrate) and its analogs on serum lipids and on the rate-limiting enzyme of the hepatic peroxisomal beta-oxidation pathway, fatty acyl-coenzyme A oxidase. Clofibrate feeding (5 g/kg diet) led to 8-10-fold increases in enzyme activity in 7 days. The enzyme remained elevated during the 28-day course of the experiment. The treatment did not lead to a lowering of serum cholesterol or triacylglycerol at any time during the experiment. In separate experiments rats were given clofibrate or one of five analogs by gastric intubation for 4 consecutive days. Four of the five analogs studied caused a significant lowering of serum triacylglycerol to about 50% of pretreatment level; another analog and clofibrate itself did not significantly affect serum triacylglycerol levels. None of the five analogs caused an induction of hepatic fatty acyl-CoA oxidase, while clofibrate treatment led to a 3-4-fold increase in enzyme activity. The results demonstrate a complete dissociation between the hypolipidemic action of these compounds and the induction of peroxisomal beta-oxidation. Thus it appears that the two phenomena are mechanistically unrelated.

Acyl-CoA Oxidase↗
PubMed1984

The use of radioactive cysteine methyl ester for labeling glycosylated molecules oxidized by periodate or neuraminidase plus galactose oxidase.

Treatment of rat lymph node cells with periodate or neuraminidase plus galactose oxidase initiates blast transformation and cell division of T lymphocytes. Either treatment introduces aldehyde functions onto glycosylated molecules of the plasma membrane. Reduction of the aldehydes with borohydride leads to a concentration-dependent inhibition of the mitogenic response. Cysteine methyl ester (Cys(Me], which can form a stable thiazolidine adduct with aldehydes, also inhibits mitogenesis in a concentration-dependent manner. Maximum inhibition is achieved at Cys(Me) concentrations about 10-fold lower than those required for borohydride (0.4 and 5 mM, respectively). [35S]Cys(Me) has been synthesized and compared with [3H]borohydride as a labeling reagent for molecules on the plasma membrane oxidized by periodate or neuraminidase plus galactose oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled whole cell lysates or of crude membrane fractions prepared from labeled cells revealed that the same oxidized molecules are specifically labeled with both reagents. Homogenates of cells labeled with either radioactive reagent were fractionated by differential and isopycnic centrifugation. The fractions were analyzed for radioactivity and for a number of marker constituents localized in various subcellular organelles. Following treatment with either reagent, the radioactivity that was covalently incorporated into macromolecules was primarily associated with sedimentable components that distributed among the fractions like plasma membrane markers. When compared with [3H]borohydride, Cys(Me) offers several advantages as a surface labeling reagent for glycosylated plasma membrane molecules, chiefly the possibility of preparing reagents labeled with isotopes other than tritium, including those like 35S, which are much stronger radioactive emitters.

Animals↗
PubMed1983

Characterization of rat lymphocyte cell membranes by analytical isopycnic centrifugation.

Lymph node cell homogenates were fractionated by differential or isopycnic centrifugation and the fractions analyzed for biochemical markers with particular focus on plasma membrane constituents. Markers for the nucleus (DNA), mitochondria (cytochrome oxidase), and lysosomes (acid hydrolases) showed the expected distributions which were different from those of membrane-bound enzymes. 5'-Nucleotidase, alkaline phosphodiesterase, gamma-glutamyltranspeptidase, and cholesterol were membrane-bound and distributed identically after isopycnic centrifugation with peaks at 1.15. The distributions of the enzymes were all shifted to higher densities by digitonin treatment, confirming their association with plasma membrane-derived elements. The distribution of galactosyltransferase (ovalbumin acceptor), largely overlapped those of plasma membrane markers but it was only slightly shifted by digitonin, suggesting its localization in Golgi apparatus. The distribution of mannosyltransferase (dolichyl phosphate acceptor) also overlapped those of plasma membrane and Golgi markers but it was centered at higher density (1.18) and was unaffected by digitonin. It is a useful marker for endoplasmic reticulum. 50% of the activity was in low speed "nuclear" sediments where it was associated with the nuclear membrane. A number of other putative and previously used markers for the endoplasmic reticulum of lymphocytes were shown not to be localized in these membranes. In particular, NADH-cytochrome c reductase was only partly associated with the endoplasmic reticulum (56%) and the remainder of the activity was in mitochondria (44%). The results show the heterogeneity in equilibrium density of plasma membrane vesicles and the considerable overlap of their distribution with those of other cellular membranes; they should provide a basis for the more rational design of preparative procedures for the lymphocyte plasma membrane.

Animals↗
PubMed1983

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity.

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.

Acyl-CoA Oxidase↗
PubMed1980

Effects of vitamin A deficiency on the levels and distribution of retinol-binding protein and marker enzymes in homogenates and Golgi-rich fractions of rat liver.

The levels of retinol-binding protein, prealbumin, and several 'marker' enzymes were determined in homogenates, crude subcellular fractions and isolated Golgi apparatus prepared from the livers of vitamin A-deficient and control rats. Vitamin A deficiency led to a marked increase (3.5-fold) in hepatic retinol-binding protein concentration and to slight increases in hepatic pre-albumin levels, without affecting the levels of a number of marker enzymes localized in various subcellular compartments. The distributions of total protein and marker enzymes among various subcellular fractions were nearly identical in control and vitamin A-deficient preparations. In particular, vitamin A deficiency had no effect on the yield or enzymatic composition of isolated Golgi-rich fractions. In vitamin A-deficient rats, where the normal secretion of retinol-binding protein was blocked, a maximum of less than 10% of the total liver retinol-binding protein was accounted for in the Golgi. In contrast, in control rats, where the secretion of retinol-binding protein was proceeding at the normal rate, the relative amount of retinol-binding protein in Golgi increased to about 23% of the total liver pool. The data suggest that the Golgi apparatus is involved in the pathway of retinol-binding protein secretion from the liver, but demonstrate that the Golgi is not the major subcellular locus for retinol-binding protein in either normal or vitamin A-deficient rats.

Animals↗
PubMed1979

Unusual properties of retinyl palmitate hydrolase activity in rat liver.

These studies report the hydrolysis of retinyl palmitate with liver homogenates and homogenate fractions from retinol-depleted rats. The studies utilized an effective in vitro assay for retinyl palmitate hydrolase (RPH) activity, in which microgram amounts of retinyl palmitate were employed as substrate, followed by the chromatographic separation and fluorescence assay of free and esterified retinol. RPH activity was maximal near pH 8 in Tris-maleate buffers, and required a bile salt for stimulation. Both cholate and taurocholate stimulated the reaction, whereas a number of other detergents tested were ineffective. The enzymatic activity showed an unusual subcellular distribution, with about 40% of total RPH activity recovered in the washed "nuclear" fraction (1,500 g pellet) and about 30--35% in the 105,000 g supernatant. This unusual distribution was not observed for marker constituents for plasma membranes, nuclei, mitochondria, lysosomes, Golgi apparatus, or endoplasmic reticulum. Despite its enrichment in the "nuclear" fraction, RPH activity was not enriched in purified preparations of nuclei or plasma membranes. Thus, RPH activity was not localized in any single, characterized subcellular structure. Another striking feature of the hepatic RPH activity was its extreme variability from rat to rat as assayed in vitro. Both the unusual subcellular distribution and the marked variability in activity were not observed for a variety of other hepatic ester hydrolase activities examined. Of ten lipid and nonlipid esters tested as substrates, only the hydrolytic activities against cholesteryl oleate and phytyl oleate correlated with, and partly resembled, RPH activity in these respects. The results suggest that the observed RPH activity is relatively specific for the hydrolysis of retinyl palmitate, and may therefore be significantly involved in hepatic retinyl ester metabolism.

Animals↗