Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.

Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.     

Circadian rhythms in the phosphorylation of rat liver histones and similar basic proteins

In the rat liver, the phosphorylation of histones is subject to a circadian rhythm. Most classes of histones, which had been obtained by polyacrylamide gel electrophoresis of nuclear HCl-extracts, exhibited maximum phosphorylation at 21.00 h and at 08.00 h. This is in correlation to maxima of RNA synthesis (23.00 h) and protein synthesis (24.00 h and 12.00 h), reported in the literature. A series of basic proteins, not as yet described, could be separated from the histones. These proteins exhibit a high extent of phosphorylation and a rhythmicity analogous to that of the histones. It is suggested that these proteins are of importance in the expression of genetic information.     

Interactions of selenium and cadmium with metallothionein-like and other cytosolic proteins of rat kidney and liver

Following injection of rats with CdCl2 and [75Se]selenite using five different protocols, the metallothionein-like proteins (MTLPs) of kidney and liver cytosols were fractionated by Sephadex G-75 gel filtration and DEAE Sephacel ion-exchange chromatography. Cd and 75Se distribution in gel-filtration elution profiles was influenced mainly by the time that elapsed between administration of these elements and by the sequence of their administration. There was no Cd redistribution to high molecular weight proteins after long-term Cd injection when rats were killed 48 hr after 75Se injection. Cd was redistributed from MTLP to high molecular-weight proteins in the liver when Cd and 75Se were injected within 1-3 hr of each other. Incorporation of 75Se into MTLP of kidney and liver was independent of Cd injection. The strength of 75Se binding of MTLP was comparable to the covalent binding of 75Se to glutathione peroxidase. Cd and 75Se did not share binding sites on MTLP. In ligand-exchange studies, 1000 ppm Cd did not displace 75Se from MTLP, but 2% 2-mercaptoethanol displaced 10% of the presumably nonspecifically bound 75Se from kidney and liver MTLP. This study provides new information regarding the apparent covalent binding of Se to low molecular-weight, Cd-containing proteins in kidney and liver.     

Regulation of rat liver phosphatidylethanolamine N-methyltransferase by cytosolic factors. Examination of a role for reversible protein phosphorylation

The nature of cytosolic factors which modulate the activity of rat liver phosphatidylethanolamine (PE) methyltransferase was investigated. The combined additions of cytosol, Mg X ATP, and NaF to incubations with rat liver microsomes produced a 1.6-fold activation of the methyltransferase at pH 9.2 and a 1.3-fold stimulation at pH 7.0. Nonhydrolyzable 5'-adenylylimidodiphosphate could not substitute for ATP, although GTP could. The activation was time dependent, stable to reisolation of the microsomes by ultracentrifugation, and partially preventable by other cytosolic components. Despite these indications that PE methyltransferase might be a substrate for cytosolic protein kinases, cAMP and Ca2+-calmodulin exerted little influence on the activation reaction. Furthermore, microsomal PE methyltransferase activity was unaffected by purified preparations of cAMP-dependent protein kinase, calmodulin-dependent protein kinase, and casein kinase II, nor was methyltransferase activity influenced by the purified catalytic subunits of protein phosphatases 1 and 2A. Cytosol also contained inhibitors of PE methyltransferase which could overcome the Mg X ATP X NaF-mediated activation of the enzyme, but were not affected by the thermostable phosphatase inhibitors 1 and 2. Part of this inhibitory activity (apparent molecular mass of 15 X 10(3) daltons) was insensitive to trypsin and chymotrypsin, stimulated by Mn2+, and partly inhibited by NaF. Therefore, regulation of methyltransferase by reversible phosphorylation, while still a tenable hypothesis, is apparently more complex than previously proposed.     

Ascorbate-Fe2+ lipid-peroxidation of rat liver microsomes: Effect of vitamin E and cytosolic proteins

In the present study, we examined the effect of the intraperitoneal administration of vitamin E (100 mg/kg weight/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of rat liver microsomes . We also analyzed the effect of hepatic cytosolic proteins on this process. The results indicate that the ascorbate induced light emission was 76% lower in microsomes (1 mg protein) obtained from vitamin E treated animals when compared with controls. In the presence of cytosolic protein (1 mg) the chemiluminescence of control microsomes diminished 55.8 and 59.5% when cytosol from controls and treated animals was used, respectively. The chemiluminescence of vitamin E microsomes diminished 25.03 and 22.08% when both types of cytosol were added to the medium. Dialyzed or treated at 70°C cytosol was also able to inhibit the lipid peroxidation of either control or vitamin E rat liver microsomes. By means of gas chromatography we analyzed the fatty acid composition of native and peroxidated microsomes from both animal groups. The peroxidation affected principally arachidonic acid and its diminution was more evident in the control microsomes than in the microsomes from the vitamin E treated group. By HPLC we analyzed the vitamin E content in all subcellular fractions employed. In microsomes from the vitamin E-group, the content of vitamin was 11 times higher than in the control ones (0.678 ± 0.1038 vs. 0.062 ± 0.0045 g -tocopherol/mg protein, respectively), while levels in the cytosol from the vitamin E-group were only 2 times higher than in the control cytosol (0.057 ± 0.0051 vs. 0.025 ± 0.0015 g -tocopherol/mg protein, respectively).     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme

A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 microM) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a pI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein.     

cAMP-dependent phosphorylation of RER proteins from rat liver: relationship with GTP-dependent membrane fusion.

Incubation of stripped rough microsomes (SRM) with the catalytic subunit of protein kinase A (PKA) permitted specific phosphorylation of seven proteins having relative molecular mass values of 55, 35, 23, 22.5, 22, 18.5 and 16.5 kDa (P55, P35 etc.). By two dimensional gel analysis, we compared these phosphoproteins with low-molecular-weight GTP-binding proteins and revealed that P23 and P22.5 co-migrated with known GTP-binding proteins. Next we examined the effect of cAMP-dependent phosphorylation on a GTP-dependent membrane function, membrane fusion. Quantitative analysis indicated no difference in the amount of membrane fusion obtained whether SRM were incubated in the absence or in the presence of PKA. Thus several rough microsomal proteins underwent cAMP-dependent phosphorylation and this post-translational modification did not affect GTP-dependent membrane fusion in a cell free system.     

Multisite phosphorylation of tau proteins from rat brain

tau proteins from adult and young rat brains were phosphorylated in vitro by protein kinases present in microtubule preparations. Several phosphates were incorporated in each molecular species of this group of proteins. Cyclic AMP dependent protein kinases and casein kinase (type I) phosphorylated tau proteins on different sites. These observations indicate that tau proteins are an example of multisite phosphorylation.     

The phosphorylation of the proteins of rat liver heterogeneous ribonucleoprotein particles by an endogenous kinase activity

An endogenous protein kinase activity of liver heterogeneous nuclear ribonucleoprotein particles has been characterized and the particle proteins which it phosphorylates in vitro have been fractionated on two-dimensional polyacrylamide gels. The activity is dependent on Mg2+ and is further stimulated by cyclic AMP, polyamines and Mn2+.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.