K.Ananth Narayan

Interaction of Ca2+ with endoplasmic reticulum of rat liver: A standardized procedure for the isolation of rat liver microsomes

Abstract A standardized method has been developed for the rapid isolation of rat liver microsomes using Ca 2+ and its advantages over other available methods have been outlined. In addition to hydrolytic enzymes and chemical composition, the important enzymes in the electron transport system were determined in Ca 2+ microsomes and normal 105,000 g microsomes and indicate only minor differences between the two preparations. Two classes of microsomes—smooth and rough particles prepared with or without the addition of Ca 2+ —were compared for their chemical and biochemical properties and indicated little differences within each microsomal fraction. The ability of other divalent cations like Mg 2+ , Fe 2+ , Ba 2+ , Zn 2+ , and Hg 2+ to aggregate the microsomes was observed while the monovalent and trivalent cations tested did not appreciably sediment the microsomes under the present experimental conditions.

A simple procedure for the isolation of rat liver microsomes

Endoplasmic reticulum of rat liver undergoes extensive fragmentation upon homogenization [l] . The major component of endoplasmic reticulum which accounted for lo-13% of total liver was recognized as microsomes in early 194 1 [2] . The rat liver microsomes were reported to have variable size from 50300 nm [3], necessitating a high centrifugal force to isolate them in contrast to other subcellular fractions like nuclei and mitochondria. The standard procedure, thus requires a sophisticated instrument like an ultracentrifuge and, at the same time, is very time consuming. Taking advantage of one of the characteristic properties of rat liver microsomes and rat skeletal muscle microsomes [4, 51 to bind calcium ions, we report in this communication a relatively simple method for the isolation of rat liver microsomes.

Lowered serum concentration of high density lipoproteins in cholesterol-fed rats

Abstract Holtzman rats were fed a solid semisynthetic diet both with and without cholesterol. The serum lipoproteins were determined by disc electrophoresis of prestained staum, preparative ultracentrifugation at several solution densities (1.006, 1.050, 1.063, 1.125, and 1.21 g/ml), disc electrophoresis of isolated lipoprotein fractions using a lipid stain as well as a protein stain, and by chemical analyses of lipoprotein proctions for their total lipid, cholesterol, phospholipid, and protein content. The data obtained have convincingly shown that, while there was an increase in serum low density lipoproteins, the serum high density lipoproteins, particularly HDL 1 , and HDL 2 were substantially decreased in cholesterol-fed rats. A speculative hypothesis for the decline in rat serum high density lipoproteins has been proposed, and, as an extension, it is further speculated that human atherosclerosis may be primarily the result of hepatic disorder, as originally suggested by Olson.

Disk electrophoresis of isolated rat-serum lipoproteins

1. 1. Disk electrophoresis was performed in 3.75% polyacrylamide gel with ultra-centrifugally isolated rat-serum lipoprotein fractions. These included the very low density, low density and two high density lipoprotein fractions. 2. 2. Four different stains, namely, sudan black B, amido black 10B, oil red O and iodine were employed and the results indicated the presence of at least seven lipo-protein components among the various fractions. Densitometry has been conducted on the gel patterns and has proved useful in the identification of the several disk-electrophoretic components. 3. 3. By comparison of the rat-serum lipoprotein patterns with those obtained with human-serum lipoproteins, it was concluded that the rat low density lipoproteins contained components which were probably smaller in size than those of the human-serum low density lipoproteins. The possibility that one of these components might represent the rat high density lipoprotein component (HDL1, d = 1.050) has been indicated. 4. 4. The principal component of rat high density lipoprotein (1.125 < d < 1.21) migrated slower than the principal component of the rat high density lipoprotein (1.063 < d < 1.125).

The effect of dietary supplementation of cholesterol and its subsequent withdrawal on the liver lipids and serum lipoproteins of chickens

Two groups of male chickens were fed either a control diet (group N) containing a standard poultry ration admixed with 10% corn oil or a cholesterol diet (group C) in which the control diet was supplemented with 1% cholesterol. After 6 weeks on the diets, a negligible amount of very low density lipoprotein (VLDL) was found in the serum from control animals. On the other hand, the serum VLDL from the cholesterol-fed birds was the predominant lipoprotein and carried 72% of the total serum lipids. Surprisingly this lipoprotein from cholesterol-fed animals was very low in triglycrides (6%) and high in total cholesterol (77%). While the level of serum low density liporotein was unaffected by the ingestion of cholesterol, the concentration of total lipids and phospholipids in the high density lipoprotein decreased in cholesterol-fed animals. The greatest change in liver lipids from animals fed cholesterol was found in the cholesterol esters, whereas the unesterified cholesterol, triglyceride and phospholipid varied slightly or remained constant. In normal animals the distribution of cholesterol between the liver and the serum was about equal, whereas in the cholesterol-fed birds the liver accounted for 80% of the cholesterol found in the liver-serum pool. In order to determine how the hypercholesterolemic bird responds to the withdrawal of cholesterol from the ration, a diet-exchange experiment was conducted. In this study the birds that were originally fed the cholesterol diets (group C) for 6 weeks were placed on the control diet (group CN) and the birds fed the control diet (group N) for 6 weeks were given the cholesterol diet (group NC). At periodic intervals, 1, 3, 7 and 14 days following the change of diets, 3–5 animals from each group were sacrificed, and analyses performed on their serum lipoproteins and liver lipids. Within one day after the diet substitution, there was a 31-fold increase and a 46% decrease, respectively, in the serum VLDL concentration in groups NC and CN as compared with their corresponding steady state values (groups N and group C, respectively). The liver cholesterol increased 4-fold and decreased 40%, respectively, in the two groups NC and CN as compared with the values obtained before the diet substitution. It is suggested that the concentration of cholesterol in the liver is the principal factor controlling cholesterol metabolism in chickens fed a hypercholesterolemic diet.

Some quantitative aspects of the disc electrophoresis of ovalbumin using amido black 10B stain

Abstract The quantitative aspects of the disc electrophoretic technique were investigated using a purified protein, egg ovalbumin. Depending on the filter used, a linear relationship between peak area and protein concentration was found up to about 40 μg of protein by densitometry. Both diffusion and gel slicing studies indicated that linearity could be extended to almost 160 μg of protein. By elution of the amido black dye from the protein-dye complex in the gel, a nearly constant dye to protein ratio was indicated. These results suggested that quantitation of the stained bands on polyacrylamide gels was limited by the nonlinear response of the densitometer, perhaps due to the nonlinearity of dye absorbance at large optical densities and not by variable amounts of dye binding to the protein bands.

Serum lipoproteins of rats bearing transplanted morris hepatoma 7777

The serum lipoprotein patterns of Buffalo‐strain rats bearing hepatoma 7777 were determined in the 2nd, 4th and 5th weeks after transplantation of the tumor. The result indicated the presence of a slow‐moving HDL component which was similar to that observed previously in Holtzman rats given 0.03% N‐2‐fluorenylacetamide. Five weeks after transplantation of the tumor, the serum lipoproteins were isolated ultracentrifugally and quantified by chemical analyses and by disc electrophoresis using a protein stain. These results have confirmed the increase in serum lipoproteins, especially the high density lipoprotein, HDL2, in these tumor‐bearing rats. The serum total lipid, cholesterol and phospholipid were considerably increased while the total serum proteins were only slightly elevated in rats with tumors as compared to control rats. A new α1‐globulin component was observed in the serum protein patterns in the tumor‐bearing rats.

Disc electrophoresis of subclasses of human serum low density lipoproteins

Serum chylomicrons, very low density lipoproteins (d < 1.006) and two classes of low density lipoproteins (1.006 < d < 1.019 and 1.019 < d < 1.050) were isolated from freshly collected normal human blood. Disc electrophoresis was performed with microquantities of these fractions using the standard spacer gel and with main gel concentrations of 1.9, 2.5, 3.0, 3.75, and 5.0%. Three different stains, namely, amido black 10B, Sudan black B and oil red O were employed for detecting the lipoprotein components in gels of 3.0, 3.75, and 5.0% concentration and sudan black B prestain was used for gels of 2.5 and 1.9% concentration. The results demonstrated that all the lipoproteins, except the chylomicrons, penetrated the standard spacer gel. In 3.0% gel, the very low density lipoproteins and the larger low density lipoproteins (1.006 < d < 1.019) were detected as separate bands behind the β-lipoprotein band. The relative migration distances in 3.0% gel of these lipoproteins were confirmed by investigating the electrophoretic behavior of synthetic mixtures of lipoproteins. The resolution obtained was superior to that previously reported with other electrophoretic methods and may find application in clinical investigations involving serum lipoproteins.

A MODIFIED DISC ELECTROPHORETIC METHOD FOR ANIMAL BLOOD SERUM PROTEINS.

Abstract A modified disc electrophoretic method has been developed and the serum electrophoretic patterns of chick, rat, mouse, guinea pig and rabbit have been obtained from micro quantities of blood. The modification involved use of sucrose solution rather than the large pore gel as the anti-convection medium and thus eliminated the problem of incomplete sample gel polymerization. The method described required as little as 4 μl of whole blood for a complete turn, was more convenient and rapid than the usual methods and may find application in animal experimentation and in clinical work.

Effect of orotic acid and cholesterol on the synthesis and composition of chicken (Gallus domesticus) serum lipoproteins

Abstract 1. 1. Chickens fed a 1% orotic acid supplement to a control diet, had similar liver and lipoprotein protein and lipid levels as those on the control diet alone. Furthermore, in vivo radioactive labelling experiments suggested that orotic acid alone did not influence the synthesis of the serum lipoproteins. In both the control and the orotic acid fed animals no label was incorporated into the serum very low density lipoproteins (VLDL). This lipoprotein fraction was almost non-existent in these 2 dietary groups. 2. 2. Addition of 1% cholesterol (group C) to the control diet of the birds induced a large increase in the VLDL concentration as well as the total cholesterol (TC) concentration of both serum and the liver. Concomitantly, a 60–70% decrease in the phospholipid (PL) along with a smaller decrease in the protein concentration of the serum high density lipoproteins (HDL) was observed. 3. 3. When 1% orotic acid was added to the cholesterol diet only about 35% of the increase over controls seen in the TC level of the VLDL and liver of group C birds was observed. Similarly, the amount of incorporation remaining in the VLDL, 4 hr after injection of label, was also about 35% of the amount that was observed in the group C chickens. 4. 4. The decline in the HDL protein and PL levels observed in the group C animals was not prevented by the addition of orotic acid to the cholesterol diet.