Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.     

The effect of levamisole and albendazole on some enzymes of Ascaridia galli and Heterakis gallinae

Ascaridia galli and Heterakis gallinae obtained from the common fowl Gallus gallus were exposed to 10(-2)-10(-5)M levamisole and albendazole; both compounds caused death of the parasites in vitro. The effect of the drugs was investigated on homogenates of the treated worms. Albendazole, at 10(-2)M, inhibited oxaloacetate reduction by 67 and 53% and malate oxidation by 21 and 17% in A. galli and H. gallinae, respectively, whereas 10(-4)M levamisole completely inhibited malate dehydrogenase activity in both directions in the two parasites. Lactate dehydrogenase was not affected significantly by either anthelmintic. Aldolase activity was diminished by 57 and 32% in A. galli and H. gallinae, respectively, with 10(-4)M levamisole. Levamisole at 10(-4)M also inhibited the activity of acid and alkaline phosphomonoesterase and cholinesterase. Albendazole had no significant effect on these enzymes in either parasite. Malate dehydrogenase and cholinesterase activity of the host tissue (intestine and caecum) was also reduced significantly with 10(-2) and 10(-3)M levamisole. These studies indicated a multiple mode of action of levamisole and albendazole.     

Mitochondrial phospholipids of rat skeletal muscle are less polyunsaturated than whole tissue phospholipids: implications for protection against oxidative stress

The fatty acid composition of phospholipids is an important determinant of membrane function. Although the mitochondria play a pivotal role in skeletal muscle function, the fatty acid composition of their individual phospholipids has not been examined. The purpose of this study was to determine the fatty acid profile of each phospholipid in rat skeletal muscle mitochondria and compare it with that of the whole muscle. Lipids were extracted from the gastrocnemius muscles of 10 Wistar rats, and phospholipids were separated by thin-layer chromatography. The fatty acid composition of each phospholipid was then determined by gas chromatography. The same procedure was applied to a mitochondrial preparation from these muscles. We found that the fatty acid composition of the individual mitochondrial phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, cardiolipin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysophosphatidyl choline) and of the total mitochondrial phospholipids differed markedly (P < 0.05) from the fatty acid composition of the corresponding whole muscle phospholipids. Notably, the mitochondrial phospholipids had higher percentages of MUFA [13.9 (2.1) vs. 10.3 (0.9)] and lower percentages of PUFA [34.8 (4.3) vs. 39.5 (5.2)] and n6 fatty acids [25.0 (2.5) vs. 27.6 (2.5)]. Overall, the mitochondrial phospholipids had a lower unsaturation index than whole muscle phospholipids [135 (20) vs. 161 (26)]. Because PUFA are susceptible to peroxidation, unlike saturated fatty acids and MUFA, we propose that the low polyunsaturation of mitochondrial phospholipids is the result of selective pressure toward membranes that are more resistant to oxidative damage by reactive oxygen species produced in their vicinity. The negative effect of the low polyunsaturation on membrane fluidity may be counterbalanced by the higher percentage of MUFA and the known low cholesterol content of mitochondrial membranes.     

Glycerolipid biosynthesis in porcine adipose tissue in vitro. I. Assay conditions for homogenates

Previous studies on glycerolipid biosynthesis in swine adipose tissue in vitro resulted in synthesis of primarily phospholipid, whereas triacylglycerol represents the vast majority of adipose tissue lipids. The objectives of this research were to maximize synthesis of triacylglycerol in vitro using the 700 x g infranatant fraction of a swine adipose tissue homogenate as the enzyme source. The capacity for total lipid synthesis was increased by greater than 50%, and the proportion of lipids synthesized as triacylglycerols was increased by increasing the length of incubation time from 20 to 60 min and the concentration of enzyme in the incubation from that obtained from 33 to that obtained from 120 mg adipose tissue. It is recommended that glycerolipid biosynthesis be assessed using two assays. An assay of up to 10 min was linear with incubation time and measured the initial incorporation of glycerol-3-phosphate into the pathway (GPAT); this incorporation was mostly into phospholipids. An assay of about 60 min was not linear with incubation time, but incorporation into total lipids (LSC) was predominantly into the triacylglycerol fraction. Although the LSC assay was not linear with time, it represents steady-state conditions that more closely typify conditions in situ. Oleate at .6 mM was inhibitory with enzyme extracted from 33 or 75 mg adipose tissue, whereas palmitate was not. Palmitoyl-CoA was not a suitable substrate because it produced low LSC and little triacylglycerol. Fluoride increased LSC but inhibited conversion of phospholipids into triacylglycerols, so its presence is not recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate of labeled choline administered intraruminally to pregnant ewes given manganese-deficient or -supplemented rations

Tissue samples were taken from pregnant ewes and their 3-month fetuses 96 hours after intraruminal dosing of the ewes with tritiated choline. Starting 5 months before mating, one group of ewes had been given a manganese (Mn)-deficient ration and a second group had been given a Mn-supplemented (normal) ration. The results indicate that Mn depletion decreased the overall uptake of choline; the radioactivity of all tissues from the Mn-depleted group was lower than the radioactivity of the corresponding tissues from the group given a Mn-supplemented (normal) ration. The radioactivity of fetal tissues was also lower in the Mn-deficient group than it was in the Mn-supplemented group. Lower radioactivity concentrations were also observed in the whole plasma (sampled at various times after radioisotope dosing) and its lipid extract of Mn-depleted ewes than in the plasma and its lipid extract of Mn-supplemented ewes. There was no significant difference in the fatty acid composition of hepatic phosphatidyl choline between the two groups. However, the proportions of linoleic, stearic, and arachidonic acids were lower in fetal hepatic phosphatidyl choline than in maternal hepatic phosphatidyl choline.     

Ascaridia galli: effect of some anthelmintics on amino acid uptake and macromolecular synthesis in vitro.

L-(U-14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine uptake by Ascaridia galli was found to be a non-linear function of time and limiting substrate concentration. The uptake was rapid initially but achieved steady state thereafter, possibly owing to the saturation of transport loci. Linear transformations of substrate saturation kinetics by Lineweaver-Burk plots of L-(U-14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine gave Kt values of 4.76, 3.03 and 2.0 mM and Jmax of 5.0, 3.57 and 2.08 m moles/100 mg dry weight/2 min, respectively. D1-tetramisole and 1-tetramisole (levamisole) inhibited the uptake of amino acids. The uptaken amino acids were readily metabolized into different tissue fractions. D1-tetramisole and levamisole significantly inhibited the incorporation of the three amino acids into the nematode's total protein, RNA and lipid fractions in an in vitro incubation system.     

氯化胆碱和乙醇胺对假丝酵母发酵棉籽饼脱毒、产酶和生物量的影响

通过测定棉籽饼发酵底物游离棉酚含量、蛋白酶活性和生物量的方法研究氯化胆碱和乙醇胺对棉籽饼发酵效果的影响。试验结果表明,发酵热处理底物时,氯化胆碱、乙醇胺均不能促进发酵脱毒,但可极显著提高底物中性蛋白酶、酸性蛋白酶活性以及底物生物量,其中氯化胆碱效果较好。发酵非热处理底物时,乙醇胺显著降低发酵脱毒效率、底物生物量,氯化胆碱有促进发酵脱毒的趋势、极显著提高底物生物量;两处理因素均可极显著提高底物碱性蛋白酶活性,降低酸性蛋白酶活性。氯化胆碱对棉籽饼底物发酵脱毒和底物营养价值有重要的意义。     

Lysine uptake by mammary gland tissue from lactating sows

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.     

In vitro studies on the synthesis of intramuscular fat in the longissimus dorsh muscle of pigs

The amount of intramuscular fat is one factor contributing to the palatability of meat. As a preliminary attempt to study the capability of M. Longissimus dorxi in pigs to synthesize intramuscular lipids, an in vitro techniques was designed. Tissue alices were incubated with glucose U-C¹⁴ or acetate-1-C¹⁴ in the presence and absence of non-radioactive glucose and insulin. Following incubation, the quantities of C¹⁴-labelled total lipids, fatty acids, and individual lipid classes, produced from each substrate were determined. The results indicate that intramuscular lipids can be synthesized in the muscle tissue, but at a slow rate. It was found that the conversion of glucose into lipids, fatty acids, and triglycerides was markedly stimulated by an increase of the glucose concentration of the medium from 2 to 5 mM. Insulin had no effect on the amount of glucose converted into total lipids. However, at both glucose concentrations insulin significantly stimulated the utilization of glucose for production of giyearide-glycerol at the expense of fatty acid synthesis. Acetate was utilized to a greater extent than glucose for synthesis of fatty acids. The presence of glucose and insulin had no effect on the utilization of acetate for production of total lipids, fatty acids, and individual lipid classes. The major part of the radioactivity of total lipids was incorporated into the triglycerides both when glucose-U-C¹⁴ and acetate-1-C¹⁴ were used as substrates.     

Effect of parbendazole and piperazine adipate on the activity of some enzymes of Ascaridia galli and Heterakis gallinae

Adult Ascaridia galli and Heterakis gallinae obtained from the fowl (Gallus gallus) were treated in vitro with 10(-2) to 10(-5) M parbendazole and piperazine adipate for 10-60 min at 38 degrees C. Both the compounds at 10(-2) M caused mortality of A. galli and H. gallinae after a maximum of 30 min exposure. The effect of the drugs on the homogenates of the treated worm was investigated. Parbendazole (10(-2) M) inhibited malate oxidation by 68% in A. galli and 62% in H. gallinae. Piperazine adipate (10(-2) M) inhibited malate oxidation by 78% in both parasites. In A. galli oxaloacetate reduction was inhibited by 41 and 26% by 10(-2) M parbendazole and piperazine adipate, respectively; with H. gallinae this inhibition was found to be 39 and 55%, respectively. Aldolase activity in both the parasites was also inhibited by 10(-2) M parbendazole and piperazine adipate. Both compounds caused an inhibition of acid phosphomonoesterase activity, but the activities of lactate dehydrogenase and alkaline phosphomonoesterase were not affected significantly. Parbendazole (10(-2) M) had no significant effect on the cholinesterase activity of these parasites, but piperazine adipate (10(-2) M) caused an inhibition of 96% in A. galli and 93% in H. gallinae. The possible mode of action of the drugs is discussed.     

Anthelmintic action—A metabolic approach (a review)

The effects of anthelmintic drugs on intermediary metabolism of parasites can be examined using techniques developed for the investigation of metabolic regulation. Most attention has been paid to the pathways of respiratory metabolism in parasites, since maintenance of high energy levels is essential to an organism's survival. Respiratory metabolism is thus a prime target for anthelmintic attack. Parasites differ from their hosts in a number of pathways of respiratory metabolism and they produce incompletely-oxidised organic acids as end products.A variety of anthelmintic drugs have been shown to inhibit some enzymes of these pathways, including phosphoenolpyruvate carboxykinase, malate dehydrogenase, and the fumarate reductase, ‘succinoxidase’ and succinate decarboxylase systems. Energy synthesis in mitochondria from parasites is also inhibited by many anthelmintics: this target is a particularly important one since its properties differ significantly from those of the host. This process requires considerable study and it may not necessarily be valid to equate ‘uncoupling’ in mammalian tissues with that in parasite tissues. Changes in ATP levels affect flux through the respiratory pathways; a complete assessment of effects of anthelminstics on the respiratory pathways of parasites would include measurement of the uptake and utilization of carbon sources (e.g., glucose and glycogen), of metabolic intermediates, including adenine nucleotides, and of excreted end products. Sites of change can then be identified and studied in greater detail.Comparison of the effect of mebendazole in vivo and in vitro on unrelated species, Moniezia expansa and Fasciola hepatica adults, sheds some light on the mechanism of its anthelmintic effects. This drug elicits similar changes in proportions and totals of adenine nucleotides in the two species, but it differs in the rapidly of its effect, its effect on glucose uptake and glycogen depletion, and in changes in respiratory end product. Consequently mebendazole may not act in the same way against these two species. It also affects a number of other systems in other parasites: the suggestion of a single ‘mode of action’ may be inappropriate for this compound.Respiratory metabolism is not the only system affected by anthelmintics: other parasite systems are also attacked, but the final effect of lethal anthelmintic is observed as changes in the respiratory metabolic pathways. Some anthelmintics, for example, levamisole, affect the respiratory pathway directly, but also affect other systems (the nervous system in the case of levamisole) and may be effective in vitro but not in vivo. In these cases it is necessary to distinguish between effects that may be subordinate to a primary effect, and to take host and permeability factors into account.     

Parasitic infections of the grey-breasted helmet guinea-fowl (Numida meleagris galeata) in Nigeria

The major helminth parasites found in wild, semi-wild and golden Sovereign stock guinea fowl were Heterakis gallinarum, Ascaridia galli, Capillaria caudinflata, Raillietina tetragona and R. echinobothrida, while Eimeria species was the most important gastro-intestinal protozoan parasite. The incidence of the latter was higher in the semi-wild stock than in the wild stock. Necropsy of dead guinea-fowl indicated that A. galli, H. gallinarum and Eimeria species were indeed responsible for their deaths, especially in the young birds. Parasites found in blood smears were Leucocytozoon sp., Plasmodium sp. and Aegyptianella pullorum. The only tick found, Argas persicus, was on a few semi-wild stock, while lice of genus Damalinia were found only on wild birds.     

Effect of Calea serrata Less. n-hexane extract on acetylcholinesterase of larvae ticks and brain Wistar rats

The stem bark of Acacia oxyphylla Graham ex Bentham is used as an anthelmintic by the natives of Mizoram (North-East India). Therefore, the aim of the study was to assess the effect of the active compound isolated from A. oxyphylla on the tegument of adult Raillietina echinobothrida and Ascaridia galli. The test parasites R. echinobothrida and A. galli were incubated in physiological buffered saline containing 0.0005, 0.001, 0.05, 0.1 and 1mg/ml of the isolated compound. The alterations in the tegument of the parasites post paralysis were examined using electron microscopes. The compound reduced the cestode's motility soon after incubation, but did not induce paralysis in the nematodes till about 11-14 h at highest concentration. The compound caused extensive digestion of cestode tegument as evident by electron microscopy. Disorganization of muscle bundles, loss of cell-cell contact, extreme vacuolization and oedema were some of the changes observed. Loss of cellular organelles combined with distortion of those present was markedly noted throughout the parasite tissue. Deformation and disorganization of epicuticle, disruption of mitochondrial and nuclear membrane were also observed in nematode exposed to the active compound of the plant. Substantial structural deformities in the treated parasites are indicative of an efficient vermicidal activity of the isolated compound against cestodes and nematodes.     

Helminth parasites of indigenous chickens in Oodi, Kgatleng District, Botswana

Thirteen adult indigenous chickens from Oodi, Kgatleng district, Botswana, were examined for helminth parasites. Two species of nematodes, Ascaridia galli and Heterakis gallinarum, and species of the cestode genus Raillietina, were recovered. A. galli and H. gallinarum were the most commonly seen parasites. The nematode A. galli occurred concurrently with Raillietina spp.     

Changes in lipids and free fatty acid fractions in adult Haemonchus contortus during incubation in vitro

Adult Haemonchus contortus (Rud., 1803) has been investigated for its ability to utilize lipids with regard to the total lipids, sterols, free fatty acids, acylglycerols and phospholipids produced during incubation in vitro. All these components exhibit extensive fluctuations, decreasing at some times and increasing at others, thus indicating both biosynthesis and utilization. Also, changes in fatty acid components of total lipids have been analyzed by gas liquid chromatography. These observations indicate that H. contortus is capable of utilizing 16:0, 18:0, 18:1, 18:2, 20:0, 20:un1, 20:un2, 21:0, 21:un1, 21:1, 21:un2, 22:un1, 22:3, 22:?, 22:un2 and 24:3 fatty acids. At the same time, because of large increases in 15:0, 16:0, 18:0, 18:?, 18:1, 18:2, 20:0, 20:un1, 20:un2, 21:0, 21:un1, 21:1 and 21:un2 acids, it is postulated that these acids are synthesized by adult H. contortus.     

Cellular uptake of valine by lactating porcine mammary tissue

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.     

The effect of a combined dietary treatment with cholesterol and cholic acid on the lipid metabolism of geese at low or high choline concentrations.

A previous study demonstrated that a dietary treatment of young geese with cholesterol and cholic acid raises lipid concentrations in the liver. The present study was carried out to investigate whether such a lipid accumulation caused by those hyperlipidemic compounds can be intensified by low dietary choline concentrations. Therefore, 38 eight-week old geese were divided into four groups of 9 or 10 animals each and received a basal diet poor in choline which consisted predominately of maize and soy protein isolate over a period of 8 weeks. Treatment factors were supplementation of diets with cholesterol and cholic acid (0 vs. 5 g of cholesterol and cholic acid each per kg) and supplementation of choline chloride (0 vs. 1.5 g/kg). Final body weights as well as carcass weights were neither influenced significantly by dietary treatment with cholesterol and cholic acid nor by low dietary choline concentrations. However, feeding diets supplemented with cholesterol and cholic acid markedly increased liver weights (two-fold), hepatic triglyceride (3.7-fold) and cholesterol (12-fold) concentrations and percentages of monounsaturated fatty acids at the expense of saturated and polyunsaturated fatty acids in the liver. In geese fed diets with cholesterol and cholic acid, insufficient choline supply did not intensify, but even slightly reduced hepatic lipid accumulation. Geese fed diets with cholesterol and cholic acid exhibited markedly increased levels of cholesterol, triglycerides and phospholipids in plasma and very low-density lipoproteins, regardless of the choline supply. Muscle tissue of geese fed diets supplemented with cholesterol and cholic acid exhibited also increased concentrations of triglycerides and cholesterol whereas the fatty acid composition of muscle lipids remained unchanged. Among geese without hyperlipidemic treatment, concentrations of triglycerides in plasma and very low-density lipoproteins as well as the concentrations of phosphatidylcholine in liver and muscle tissue were not reduced by low dietary choline concentrations. Therefore, it is suggested that those animals were able to synthesize endogenous sufficient choline.     

In vitro effects of insulin on glucose and lipid metabolism in rat embryos

Glucose is utilized for oxidation and synthesis of various lipids in cultured rat embryos. The present experiment examined the effect of insulin on the incorporation of glucose into lipid fractions in rat embryos in vitro . Embryos at the 2-cell, 8-cell and blastocyst stages were incubated for 5 h in hamster embryo culture medium (HECM)-1 containing ¹⁴C-glucose and 170 nmol/L insulin, or in HECM-1 containing only ¹⁴C-glucose, and the oxidation of glucose in these embryos was examined. In addition, the total lipids of blastocysts were separated by thin layer chromatography and the radioactivity of the separated lipid fractions was measured. Oxidation of glucose was significantly increased after insulin treatment compared with that without insulin treatment in 8-cell embryos and blastocysts ( P  < 0.05), but not in 2-cell embryos. Incorporation of glucose into lipids in blastocysts was significantly lowered by insulin treatment compared with that without insulin treatment ( P  < 0.05). Most of the radioactivity was recovered from triacylglycerols of blastocysts and the remaining radioactivity was found in other neutral lipids and phospholipids. We conclude that insulin accelerates the utilization for oxidation of glucose and inhibits the storage of triacylglycerols in rat blastocysts.     

Glycerolipid biosynthesis in porcine adipose tissue in vitro. II. Synthesis by various types of cellular preparations

Glycerolipid biosynthesis by porcine adipose tissue homogenates did not yield the 90+% triacylglycerol observed in situ. Consequently, we compared intact tissue slices and various subcellular fractions to characterize the usefulness of such systems to assess glycerolipid biosynthesis in vitro. Glycerolipid biosynthesis by porcine adipose tissue homogenates was measured in vitro using either [14C]-fatty acid or [14C]-glycerol-3-phosphate (G3P) as a radiolabelled substrate. Removal of residual 14C-labelled fatty acid from lipid extracts was difficult. Because G3P is soluble in water, residual [14C]-G3P separated easily from the glycerolipid-containing organic phase and, thus, was the preferred radiolabelled substrate. With tissue slices, glycerol and G3P were minimally incorporated into lipid so that [14C]-fatty acid was the preferred radiolabelled tracer. A washing procedure followed by thin layer chromatography was devised to separate residual [14C]-fatty acid from glycerolipids, including phospholipids. Fatty acid esterification into glycerolipids in tissue slices yielded about 4% phospholipids, whereas with homogenates, esterification yielded up to 50% phospholipids. Comparison of several subcellular fractions indicated that microsomes contained most of the glycerolipid biosynthetic activity when activity was expressed on a protein basis. However, when activities were expressed on a tissue wet weight basis, the 700 x g infranate and the 10,000 x g supernate had about equal activity that was far greater than the microsomes. The 700 x g infranate was the preferred enzyme preparation for assay of the entrance of G3P into the pathway as well as the capacity to synthesize triacylglycerol. Several methods of freezing and storing tissue or 700 x g infranates were not acceptable. Freezing of the 700 x g infranate in liquid N2 with storage at -80 degrees C may be an acceptable procedure.