Orotic acid added to casein, but not to egg protein, soy protein, or wheat gluten diets increases 1,2-diacylglycerol levels and lowers superoxide dismutase activities in rat liver.

Effects of the dietary addition of orotic acid to a diet containing casein as a sole protein source on lipid levels in the liver and serum, activities of antioxidant enzymes in the liver, and some enzyme activities in serum, were compared with other diets containing egg protein, soy protein, or wheat gluten, respectively. 1. The contents in the liver of each lipid were increased by the addition of orotic acid as compared with those values without it. The orotic acid added to the casein diet caused accumulation of more liver total lipids, triacylglycerol, 1,2-diacylglycerol, and phospholipids than those fed three other diets. 2. The addition of orotic acid to the casein, but not to the other three diets, lowered the activities of liver superoxide dismutase and increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, the significant increase in serum ornithine carbamoyltransferase activities as the marker of liver lesions may result from the marked accumulation of liver lipids, decreased activities of hepatic superoxide dismutase, and the increased level of hepatic 1,2-diacylglycerol, followed by possibly the increased level of superoxide anion and increased activity of protein kinase C in rats fed the casein diet with orotic acid added.     

Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in native membranes.

Casein kinase I activity is present in cells as a cytosolic and a membrane-bound enzyme. Previously, the erythroid membrane-bound casein kinase I was shown to associate with purified integral membrane proteins; this association and protein kinase activity was regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet, C.E., Brockman, J.L., Lewis, D., Chan, C., and Anderson, R.A. (1990) J. Biol. Chem. 265, 7369-7376). Here we show that both the membrane-bound and the cytosolic casein kinase interact with native membranes and that this interaction is regulated by the membrane content of PIP2. On native membranes, casein kinase I activity is potently inhibited by small increases (10-20%) in the membrane content of either exogenously added or intrinsic PIP2. However, the majority of the intrinsic content of PIP2 in isolated membranes does not inhibit casein kinase, suggesting that this PIP2 is not accessible. Regulation of the casein kinases on membranes is sensitive to detergents and to chymotrypsin treatment of membranes.     

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.     

Hepatic plasma membrane fluidity and dietary proteins.

Effect of protein deficient diet on hepatic plasma membrane fluidity has been studied in rats using (i) steady state fluorescence polarization and anisotropy, (ii) phospholipid and cholesterol contents, (iii) phospholipid fatty acid composition, (iv) turnover of phosphatidyl choline (PC), and (v) activities of membrane-bound enzymes as parameters and rats fed casein (20%) diet as standard group. A significant increase in steady state fluorescence and anisotropy values was registered in the deficient group, indicating increased resistance and hence decrease in fluidity of the plasma membrane. Supplementation of the diet with lysine and threonine improved these values, thereby suggesting the significance of diet for membrane fluidity. Simultaneous significant alterations in other parameters, viz. (i) decrease in PC, PE and free cholesterol and increase in esterified cholesterol contents, (ii) decrease in unsaturation of fatty acids of PC, (iii) decrease in incorporation of NaH2 32PO4, [CH3-14C]choline and [CH3-14C]methionine into plasma membrane PC, and (iv) decrease in activities of plasma membrane 5'-nucleotidase and phosphodiesterase along with increase of (Na(+)-K+)ATPase and adenyl cyclase, were observed in the deficient group which on supplementation with lysine and threonine showed improvement over alterations.     

High-Casein Diet Suppresses Guanidinoacetic Acid-Induced Hyperhomocysteinemia and Potentiates the Hypohomocysteinemic Effect of Serine in Rats

To determine the effect of dietary protein level on experimental hyperhomocysteinemia, rats were fed 10% casein (10C) and 40% casein (40C) diets with or without 0.5% guanidinoacetic acid (GAA) for 14 d. In addition, rats were fed 10C + 0.75% methionine (10CM) and 40C + 0.75% methionine (40CM) diets with or without 2.5% serine for 14 d to determine the relationship between the dietary protein level and intensity of the hypohomocysteinemic effect of serine. GAA supplementation markedly increased the plasma homocysteine concentration in rats fed with the 10C diet, whereas it did not increase the plasma homocysteine concentration in rats fed with the 40C diet. Although serine supplementation significantly suppressed the methionine-induced enhancement of plasma homocysteine concentration, the decreased plasma homocysteine concentration was significantly lower in rats fed with the 40CM diet than in rats fed with the 10CM diet. The hepatic cystathionine β-synthase and betaine-homocysteine S-methyltransferase activities were significantly higher in rats fed with the 40C or 40CM diet than in rats fed with the 10C or 10CM diet, irrespective of supplementation with GAA and serine. These results indicate that the high-casein diet was effective for both suppressing GAA-induced hyperhomocysteinemia and potentiating the hypohomocysteinemic effect of serine, probably through the enhanced activity of homocysteine-metabolizing enzymes.     

Effects of soy protein diet on the expression of adipose genes and plasma adiponectin.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.     

Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet.

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

Comparative study of the effects of 2 types of protein-energy malnutrition followed by a balanced refeeding, on the lipase, phospholipase A2 and amylase activities of the pancreas and pancreatic juice in the growing rat

The intake of 2 p. 100 casein diet as only protein source decreased overall phospholipase A2 activity. Amylase activity was more affected than lipase one. The effects of intake of 5 p. 100 gluten diet as only protein source were less important than 2 p. 100 casein diet. Refeeding on 20 p. 100 or 15 p. 100 casein diet caused a considerable increase of phospholipase A2, lipase and amylase activities.     

Dietary orotic acid affects antioxidant enzyme mRNA levels and oxidative damage to lipids and proteins in rat liver

We investigated the effects of the dietary addition of orotic acid on liver antioxidant enzymes, mRNA levels of these enzymes, and peroxidative products by comparing casein with soy protein as the source of dietary protein. Rats fed the casein diet accumulated more liver lipids than those fed the soy protein diet when orotic acid was added. The addition of orotic acid lowered both the activity of liver Cu, Zn-superoxide dismutase and the level of Cu, Zn-superoxide dismutase mRNA. The addition of orotic acid led to a significant increase in the contents of conjugated dienes and protein carbonyls in the liver. In addition, dietary soy protein protected the increase in the levels of lipids and proteins peroxide induced by orotic acid. The addition of orotic acid to the casein diet increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, liver damage might result from the increased superoxide anion due to the decrease in the activity of hepatic superoxide dismutase, as well as increase in the production of hepatic peroxidative products in rats fed the casein diet with orotic acid.     

In vivo progesterone regulation of protein phosphatase activity in Xenopus oocytes

Exogenous beta casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in vivo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/min/oocyte, respectively, for beta casein phosphorylated by casein kinase II and beta casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of beta casein.     

Erythroid membrane-bound protein kinase binds to a membrane component and is regulated by phosphatidylinositol 4,5-bisphosphate

In the erythrocyte, a membrane-bound serine/threonine protein kinase (a casein kinase) has been shown to phosphorylate a number of membrane proteins, modulating their function. Here we report that the membrane-bound protein kinase binds to membranes by an association with a minor membrane component contained in preparations of glycophorin (possibly a minor glycophorin). The binding of the kinase to glycophorins does not significantly modify kinase activity. However, upon binding, the kinase activity is potently inhibited by phosphatidylinositol 4,5-bisphosphate, and the affinity of the kinase for the glycophorins is increased. Other phospholipids or polyanions such as inositol 1,4,5-trisphosphate or 2,3-diphosphoglycerate do not affect protein kinase activity when the kinase is bound to membranes but do inhibit the solubilized membrane-bound kinase. In the erythrocyte, there is a cytosolic form of the casein kinase which is very similar, having the same molecular weight and substrate specificity as the membrane-bound casein kinase. The cytosolic casein kinase is inhibited by 2,3-diphosphoglycerate but much less so by glycophorin preparations containing phosphoinositol 4,5-bisphosphate. When the sequences of both casein kinases were compared by two-dimensional peptide mapping, it was found that the two kinases were very similar but not identical.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

Choline deficiency activates phospholipases A2 and C in rat liver without affecting the activity of protein kinase C

There is evidence to suggest that liver tumor promoters exert their effect through the interference of signal transduction in hepatic cells. Both phospholipase A(2) and phospholipase C play important roles in the generation of second messengers and in the activation of Ca(2+), phospholipid-dependent protein kinase C. Using male Sprague-Dawley rats, we investigated whether liver tumor-promoting regimens of a choline-deficient diet and phenobarbital alter the activities of phospholipase A(2) and phospholipase C in the liver, and extended the study to determine the effect of a choline-deficient diet on protein kinase C activities. Feeding a choline-deficient diet for 1 week increased the activities of both phospholipase A(2) (50%) and phospholipase C (22%), and the activities of both enzymes were more than doubled after 4 weeks. Feeding a phenobarbital diet resulted in a slight decrease in phospholipase A(2) activities at 4 weeks but no significant changes in PLC activities. The protein kinase C activities and its distribution between soluble and particulate fractions remained unchanged after 1, 2, and 4 weeks feeding of a choline-deficient diet. Thus, activation of both phospholipase A(2) and C is distinct for a choline-deficient diet, not shared by phenobarbital diet. Increased activities of these enzymes were not associated with the activation of protein kinase C under the present experimental condition.     

Inhibition of casein kinase type 2 activity by formation of complexes with mRNA in vivo

It is known that casein kinase 2 possesses, besides the protein kinase, an RNA-binding activity. Using ligand blotting it has been demonstrated that the both activities are localized on the alpha- and alpha'-subunits of the enzyme. Casein kinase 2 is suppressed in vitro by polyuridylic acid. A part of the intracellular pool of casein kinase 2 is found in the informosomes. The informosomes and free proteins were separated by centrifugation in a sucrose density gradient, and each fraction was incubated with casein and [gamma-32P]ATP. The informosome-bound protein kinase is completely inhibited, while the free protein kinases heavily phosphorylate casein. It is concluded that the activity of casein kinase 2 can be regulated by the reversible formation of complexes with RNA.     

Threonine Entry into Rat Brain After Diet-Induced Changes in Plasma Amino Acids

Abstract: Passage of amino acids across the blood-brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/ day for 7–10 days to diets containing 6, 18 , or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18 , or 50% casein. After 4–7 h, they were anesthetized and infused intravenously with [¹⁴C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 m M , when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 m M , occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.     

Effects of protein level, methionine supplementation and carbohydrate type of the diet on liver lipid and plasma free threonine contents in the lactating rat

Eight groups of 13-15 female rats were fed purified diets after littering. Four groups received a low protein (8% casein) diet (groups 8) and the others, a normal protein (20% casein) diet (groups 20). Carbohydrates were supplied either as starch (groups S) or as starch plus 40% fructose (groups F). Half the animals received a 0.4% methionine supplementation (groups M). Four or five dams per group were sacrificed on days 2, 7 and 14 after littering. The diet intake was increased by methionine supplementation, substitution of starch for fructose and increased protein content, mainly during the second week of lactation. This influenced weight variation of the dams and litter growth. On all days, the plasma levels of cholesterol esters, triglycerides and phospholipids were positively correlated with the dietary protein level. On days 7 and 14, the liver neutral lipid content was increased in rats fed the low protein diets supplemented with methionine (groups 8SM and 8FM) and the normal protein diets containing 40% fructose (groups 20F and 20FM). The plasma free threonine content was positively correlated with the protein level in the diet. On day 14, rats fed a low protein diet had a threonine deficiency, except those in groups 8S and 8F. The plasma free threonine content of these rats was not reduced, possibly due to an impaired utilization of this amino acid. The liver lipidosis observed during lactation, in contrast to that observed during growth with a low protein diet, was not due to a threonine deficiency.     

Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.