Addition Of Deoxycholate Research Articles

Triple Lipid Screening Test: A Homogeneous Sequential Assay for HDL-Cholesterol, Total Cholesterol, and Triglycerides

The analysis of lipids in serum lipoprotein fractions is useful in assessing the risk for coronary artery disease, but it typically involves performing multiple tests. An automated single-tube assay, referred to as the triple lipid screening (TLS) test, can be used for measuring HDL-cholesterol (HDL-C), total cholesterol, and triglycerides (TGs) with no specimen pretreatment. The first part of the assay is based on a homogeneous assay for HDL-C that uses either an anti-apolipoprotein B antibody (TLS-A test) or a polyanion (TLS-B test) that blocks the enzymatic measurement of cholesterol on the non-HDL fraction. After the addition of deoxycholate, which solubilizes the unreacted cholesterol from the non-HDL fraction, the remaining cholesterol in the sample is subsequently measured enzymatically. Using the same enzyme detection system as the cholesterol assay, TGs are measured in the last step, after the addition of the enzymes for the TG assay. The TLS assay (y) had acceptable analytic performance and compared favorably with standard tests (x) for each analyte: for HDL-C, TLS-A = 0.99x + 0.19 (R = 0.980); TLS-B = 1.00x - 0.15 (R = 0.974); for total cholesterol, TLS-A = 1.03x + 0.12 (R = 0.997); TLS-B = 1.07x - 0.30 (R = 0.965); and for TGs, TLS-A = 1.02x + 0.02 (R = 0.988); TLS-B = 1.04x - 0.28 (R = 0.980). The TLS test is a single-tube homogeneous assay for the analysis of all of the major serum lipoprotein fractions and can be used as a simple screening test for the detection of hyperlipidemia.

Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0.5 mM) to the apical side induced the activation of amiloride-sensitive Na+ channels and an increase in the unidirectional Cl- fluxes. Here we used two established cell lines (A6 and Caco2) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In A6 cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110.3 +/- 4 pS pF-1 (N = 8) which was reduced to 89 +/- 14 pS pF-1 (N = 8) by the addition of DIDS (0.5 mM), The absolute values of these two effects were not statistically different (P < 0.2). In Caco2 cells, the addition of DOC (0.5 mM) induced, after 1 min, an increase in cell conductance of 583 +/- 16 pS pF-1 (N = 8) which was reduced to 560.4 +/- 16 pS pF-1 (N = 8) by DIDS (0.5 mM) and N-phenylanthranilic acid (DPC; 0.5 mM). The two values were not statistically different (P < 0.4). In Caco2 cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 +/- 64 pS pF-1 (N = 6). Subsequent addition of amiloride (0.1 mM) reduced the cell conductance to 1558 +/- 33 pS pF-1 (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0. 05. In cells in which DOC produced a conductance increase of 1010 +/- 10 pS pF-1, gadolinium (0.5 mM) induced a fall in cell conductance of 1800 +/- 10 pS pF-1. In Caco2 cells, addition of DOC (0.5 mM) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0. 5 mM) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27.7 +/- 1.9 pS and 0.8 +/- 5.7 mV, respectively. In cell-attached patches (N = 13) these values were 24.9 +/- 4.4 pS and -18.1 +/- 6.4 mV. In excised inside-out patches from Caco2 cells, subjected to electrochemical gradients for Na+, K+ and Cl-, (+85, -85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.

Effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis (Trematoda: Opisthorchiidae) metacercariae in vitro

The effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis metacercariae were studied. The velocities of metacercarial excystation, digestion of the metacercarial outer layer and tryptic BAEE hydrolysis were measured in various media. Pretreatment with acid or acid pepsin accelerated the excystation, but was not required under adequate tryptic activity. Taurocholate and deoxycholate evoked no excystation by themselves. However, in the trypsin medium with taurocholate and deoxycholate, they accelerated both the velocity of metacercarial excystation and of digestion of the outer layer, although the accelerative effect did not increase above 4 −2% deoxycholate. Taurocholate below 1% and deoxycholate below 4 −2% did not affect the rate of tryptic hydrolysis, while this was depressed by deoxycholate over 4 −2%. Addition of deoxycholate over 4 −2% also depressed the final percentage of excystation, as well as worm motility. By themselves, cysteine and 2-mercaptoethanol (MCE) swelled and deformed the cyst wall and evoked metacercarial excystation without trypsin, however, ascorbate and Na dithionite evoked no excystation. Addition of ascorbate to the trypsin medium did not change the velocity of excystation, but this was completely inhibited by the addition of dithionite and was accelerated by the addition of cysteine or MCE. Although excystation was accelerated with an increase of cysteine concentration, this acceleration was depressed at over 0.01 M cysteine. The rate of tryptic hydrolysis was depressed at over 0.002 M cysteine. The present study suggests that the excystation facilitative effects of pretreatment of acid pepsin, bile acids and reductants (cysteine and MCE) in trypsin medium are due to denaturation of protein, detergent action and cleavage of SS-linkage, respectively, on the digestion step of the outer layer of the metacercariae and cessation of the facilitative effect at a high concentration of deoxycholate is related to the rupturing step of the inner layer by larval movement.

Biochemical and pharmacologic characterization of a phosphatidylinositol-specific phospholipase C in rat neutrophils.

The phosphatidylinositol (PI)-specific phospholipase C (PLC) activity contained in sonicates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15-20 min at 37 degrees C and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5-6.0, is enhanced 1.5-3-fold by the addition of deoxycholate, and is Ca++ dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 +/- 85 microM and a Vmax of 34.3 +/- 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 +/- 66 microM and a Vmax of 14.3 +/- 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values less than or equal to 100 microM) inhibitors of the PMN PLC.

An immunoenzyme quantitative assay for the antiviral effect of interferons

A technique is described for measurement of the antiviral activity of interferon by an immunoenzymatic assay for viral proteins. Cells treated by tested samples of interferon (IFN) are infected with vesicular stomatitis virus (VSV) and following the development of viral cytopathy are lysed by the addition of deoxycholate and then transferred into ELISA microplates. The viral proteins bind effectively to the microplates proportionally to their level in the culture and may be measured by incubating the plates sequentially with (1) rabbit antiserum against VSV, (2) a conjugate of alkaline phosphatase either to protein A or to an antibody against rabbit IgG and (3) p-nitrophenylphosphate. This procedure may be further simplified by using antibodies against VSV to which alkaline phosphatase has been directly conjugated. We found this immunoenzyme assay to be superior to the ‘cytophatic effect inhibition’ assay in precision and sensitivity and in being independent of the effectiveness of viral cytopathy.

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b- c 1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b- c 1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V max and the K m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.

Addition of deoxycholate in electroimmunoassay and crossed immunofocusing for quantification of β2-glycoprotein I and its subfractions

β 2-GlycoproteinI was shown to be a hydrophilic protein exhibiting no charge shift in the presence of a cationic detergent, but a charge shift in the presence of an anionic detergent. The latter was suggested to be caused by a binding of β 2-glycoprotein I to deoxycholate in the Triton X-100/deoxycholate micelles. Quantification by electroimmunoassay of asialo- β 2-glycoprotein and I and subfractions of β 2-glycoprotein I gave different results although identical results were obtained in single radial immunodiffusion. Addition of 0.2% (w/v) of deoxycholate to the agarose gels containing Triton X-100 prior to electrophoresis, however, eliminated these differences. The effect of deoxycholate on the rate of migration of β 2-glycoprotein I was found applicable for electroimmunoassay of the protein. Crossed immunofocusing of plasma from individual donors, electrophoresed in an agarose containing Triton X-100/deoxycholate micelles confirmed a postulated variation in the relative composition of subfractions of β 2-glycoprotein I in plasma earlier suggested by Finlayson and Mushinski (Finlayson, J.S. and Mushinski, J.F. (1967) Biochim. Biophys. Acta 147, 413–420).

Disposition of gangliosides and sialosylglycoproteins in neuronal membranes.

Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[3H]N-acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H]sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 M-KCl, 125 mM-EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of th neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labelled in vivo by an intracerebral injection of N-[3H]acetylmannosamine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with behaviour of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.

Activation of alkyldihydroxyacetone phosphate synthase by detergents

The microsomal enzyme alkyldihydroxyacetone phosphate synthase, which synthesizes alkyldihydroxyacetone phosphate from fatty alcohols and acyldihydroxyacetone phosphate, was examined in the harderian gland and Ehrlich ascites carcinoma. Although the enzyme possessed a higher specific activity in the harderian gland than in the Ehrlich ascites carcinoma, similar results were obtained using the enzyme from both sources. Alkyldihydroxyacetone phosphate synthase was stimulated approximately 50% by the addition of deoxycholate or Triton X-100 to the assay system. The concentration of detergent needed to elicit this response was below the critical micellar concentration of these detergents. The reaction rate as a function of pH was essentially constant in the absence of detergent, but, when Triton X-100 was added to the system, the synthase showed a distinct pH optimum of 8.0–8.5. Unlike cytochrome b 5, a marker for the surface of the microsomal vesicles, the alkyldihydroxyacetone phosphate synthase activity was not removed from microsomal preparations treated with trypsin. Also, alkyldihydroxyacetone phosphate synthase was not solubilized by treatment of the microsomes with deoxycholate concentrations below the critical micellar concentration of this detergent. However, enzymatic activity was efficiently removed from microsomes treated in combination with deoxycholate and trypsin. We suggest from these data that the catalytic portion of alkyldihydroxyacetone phosphate synthase is exposed to the luminal surface of the microsomal vesicles. Since both dihydroxyacetone phosphate and glyceraldehyde 3-phosphate were inactive in the assay system in the presence and absence of detergent, the possibility that acyldihydroxyacetone phosphate functions in the system as a lipophilic molecule that facilitates the transport of a triose phosphate into the microsomal lumen is unlikely.

Membrane effects on drug monoxygenation activity in hepatic microsomes

The temperature dependence of drug monooxygenation in phenobarbital-induced rat liver microsomes has been investigated. With 7-ethoxycoumarin as a substrate the activity of the microsomes could be measured down to 0°C by the increase in fluorescence of the dealkylated reaction product 7-hydroxycoumarin (umbelliferone). Arrhenius plots of the activities at various temperatures between 0°C and 45°C showed a break in the activation energy around 20°C. Addition of deoxycholate or high concentrations of glycerol, known to solubilize membrane-bound enzymes, abolished the break of the activation energy. Cholesterol, incorporated into the microsomal membrane in amounts equimolar to the microsomal phospholipid content led to a decrease of the activation energy at low temperatures and to an increase at higher temperatures, resulting in a loss of the break. The activity of microsomal NADPH-cytochrome c reductase with the water-soluble electron acceptor dichlorophenolindophenol showed no discontinuity in the Arrhenius plot. In addition the cumene hydroperoxide-mediated and cytochrome P-450- dependent O-dealkylation of 7-ethoxycoumarin proceeded without a break in the activation energy. It is concluded that phospholipid phase transitions affect the electron transfer from the reductase to cytochrome P-450.

Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division.

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp2 ts 42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese's (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-14C into RNA still con-tinued even after the incorporation of N-acetyl-3H-D-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver

The phospholipases A 2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.

Comparison of rat liver and Escherichia coli cell-free systems in inhibition of polypeptide synthesis by deoxycholate

1. Polyphenylalanine synthesis by rat liver and Escherichia coli ribosomes was not affected by the addition of non-ionic detergents (Tween 40, Brij 58, and Triton X-100), while the addition of deoxycholate (DOC) produced strong inhibition. On the other hand, the rat liver polysomes prepared with the use of DOC showed no significant difference in either activity or yield as compared with those prepared with non-ionic detergents. 2. In the case of rat liver, S100 fraction was inactivated irreversibly by DOC, while no irreversible inactivation by DOC was observed with ribosomes. With respect to the components of S100 fraction, it was observed that phenylalanyl-tRNA synthetase and the elongation factor(s) (EF1 and/or EF2) were both inactivated irreversibly by DOC. 3. In the case of E. coli ribosomes, slow but irreversible inactivation by DOC was observed. E. coli S100 fraction was not inactivated irreversibly by DOC, but phenylalanyltRNA synthetase in E. coli S100 fraction was found to be inhibited reversibly by DOC. 4. Non-enzymatic poly(U)dependent binding of phenylalanyl-tRNA to ribosomes was inhibited by DOC in both rat liver and E. coli systems.

A method for the determination of lipoprotein lipase in postheparin plasma and body tissues utilizing a triolein-coated Celite substrate

A simple and specific method for assaying lipoprotein lipase activity is described. Postheparin plasma, heart homogenates, or extracts of acetone powder of adipose tissue were incubated with a triolein-coated Celite substrate, and enzyme activity was determined from the rate of free fatty acid (FFA) release in the incubation system. FFA release was linear for 30 min, and was proportional to protein concentration in the incubation system. FFA release was decreased by addition of deoxycholate or Triton X-100. Increasing the concentration of heparin in the incubation system caused a gradual decrease in FFA release by postheparin plasma and increases in activity of heart homogenates and adipose tissue lipoprotein lipase. The Celite substrate was found to be satisfactory for assaying pancreatic lipase activity as well.

Metabolism of ethanol by rat liver microsomal enzymes

In confirmation of other reports, rat liver microsomes were found to oxidize ethanol to acetaldehyde in vitro in the presence of NADPH and oxygen, at an optimal pH of 7·0–7·4. This activity was inhibited markedly by addition of deoxycholate in vitro, cyanide and azide, but only weakly by digitonin and not at all by pyrazole or SKF 525-A (β-diethylaminoethyl-diphenylpropyl acetate). 3-Amino-1,2,4-triazole inhibited the activity when added in vitro or pre-injected in vivo. Hydrogen peroxide and barium peroxide were effective in place of NADPH and oxygen when initial reaction rates were considered: this activity was also inhibited by cyanide and by aminotriazole. It is concluded that oxidation of ethanol by hepatic microsomes in vitro is brought about by a combination of catalatic activity plus a system which generates H 2O 2 from the oxidation of NADPH, rather than by the intact mixed-function oxidase (drug-metabolizing) system.

Studies of the particulate and soluble aspartate aminotransferases in germinating pea cotyledons

The soluble and particulate aspartate aminotransferases (E.C. 2.6.1.1) present in germinating pea cotyledons were isolated by differential centrifugation and partially purified by ammonium sulphate fractionation and gel filtration. The activities of both enzymes varied during the first 5 days of germination, the soluble enzyme accounting for more than 90 per cent of the total extractable aspartate aminotransferase activity. Particulate enzyme activity was solubilized by various treatments including osmotic shock, sonication and by addition of deoxycholate. The general catalytic properties of both enzymes were compared and kinetic experiments suggested that both have a binary mode of action. The ability of various vitamin B 6 derivatives to reconstitute the soluble apoenzyme demonstrated that only pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate were effective. These latter compounds were both tightly bound in the reconstituted enzyme as prolonged dialysis did not appreciably alter the specific enzyme activity. Studies of the rates of reconstitution of the soluble apoenzyme with these coenzymes showed that pyridoxal-5′-phosphate activated the enzyme more readily than equimolar amounts of pyridoxamine-5′-phosphate.

Force measurements in skinned muscle fibres.

1. Isometric force was measured in skinned segments of frog semitendinosus muscle fibres exposed to solutions in which the calcium ion concentration was controlled with EGTA.2. The threshold for force development, calculated from an apparent stability constant for the CaEGTA complex of 10(6.69)M(-1) at pH 7.0, was generally close to pCa 7.5. Maximum force was reached at about pCa 6.0.3. Maximum force is proportional to the cross-sectional area of the fibres.4. The rate of force development was slower than that expected from simple diffusion of a substance from the bathing solution into the fibre. The delay appears to be due to slow equilibration of the EGTA buffer system during calcium uptake by the sarcoplasmic reticulum.5. Addition of deoxycholate (DOC) to the bathing solution produced a reversible increase in the rate of force development. The steady force was also increased for values of pCa that gave less than maximum force, which shifted the force-pCa relation toward lower calcium concentrations by about 0.5 pCa unit.6. The length-force relation in partially activated preparations is similar to that reported for electrically activated intact fibres. This result suggests that in the region of myofilament overlap the affinity of the binding sites for calcium is uniform along the length of the calciumbinding myofilament.

Effect of Dietary Tryptophan on Hepatic Polyribosomes and Protein Synthesis in Fasted Mice

Abstract Mice were fasted overnight, and the following morning the the animals were tube-fed (a) a complete amino acid mixture, (b) the same mixture devoid of one essential amino acid (tryptophan, threonine, isoleucine, or methionine), (c) a single essential amino acid (l-tryptophan, l-threonine, l-isoleucine, or l-methionine), or (d) nothing. All animals were killed 1 hour after the feeding. Postmitochondrial supernatants of liver homogenates were prepared and used for determination of protein synthesis in vitro, or, after addition of deoxycholate, for direct sucrose density gradient analyses. The polyribosome patterns of the livers of all the groups fell generally into one of two basic patterns: A, a shift of polyribosomes toward lighter aggregates, particularly monosomes, and a concomitant decrease of heavier aggregates, or B, a shift toward heavier aggregates with fewer monosomes. All groups fed l-tryptophan alone or amino acid mixtures which contained tryptophan, whether complete or incomplete with respect to other amino acids, showed the B polyribosome pattern, while the fasted controls, the tryptophan-devoid group, or the other single amino acid groups had the A pattern. The A pattern was associated with a low level of incorporation of 14C-leucine or 14C-phenylalanine into protein in vitro, while mice with the B pattern had a considerably enhanced level of incorporation. Four milligrams of l-tryptophan, but not equimolar amounts of single compounds structurally related to tryptophan, induced an increase in stimulation of hepatic protein synthesis, which changed little with higher levels of tryptophan. l-Tryptophan rapidly reached the liver and induced enhanced hepatic protein synthesis within 5 to 10 min, reached its peak effect by 1 hour, then began to decrease within 2 hours, and returned to control fasted levels within 4 to 6 hours. Animals fasted for longer periods (40 and 64 hours) rather than overnight (16 hours), adrenalectomized mice, and mice pretreated with actinomycin D, when tubefed l-tryptophan, showed enhanced hepatic protein synthesis as well as the B polyribosome profile response. Incubation in vitro with microsomal and supernatant fractions revealed results similar to those found with postmitochondrial supernatants and indicated that the microsomes of the tryptophan-fed animals accounted for the main increase in hepatic protein synthesis. Administration of 14C-leucine intraperitoneally to tryptophan-fed mice revealed a small but significant increase in hepatic protein synthesis in vivo over that in control fasted mice. The results indicate that dietary tryptophan plays a special role in the regulation of polyribosome aggregation and protein synthesis in the liver.

The epoxidation of aldrin by housefly microsomes and its inhibition by carbon monoxide

Housefly ( Musca vicina) microsomes contain a system which converts aldrin to dieldrin and which requires the presence of NADPH and O 2. The pH optimum is 8·2 and the reaction is stimulated by cyanide and inhibited by EDTA, sesamex and carbon monoxide. The inhibition by carbon monoxide is partially reversed by light. Carbon monoxide combines with the reduced microsomes to give a difference spectrum with a peak at 450 mμ which shifts to 420 mμ on addition of deoxycholate. The possibility of the presence of two terminal oxidases is discussed briefly.