In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Selenium and selenoproteins in the rat kidney

Kidney tissue contains a high concentration of selenium that is not accounted for by the known selenoprotein glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9). In order to investigate the nonglutathione peroxidase selenium, rats were isotopically labeled with [75Se]selenite over a 10-day period. After this time half of the 75Se in kidney homogenate was found in the particulate subcellular fractions. The kidney lysosomes contained unusually high levels of 75Se, yet they did not contain correspondingly high levels of glutathione peroxidase activity. Two selenoproteins having molecular weights less than 40 000 were resolved by gel filtration from a kidney supernatant fraction. A third selenoprotein exhibited a molecular weight of 75 000. This protein contained one 75 000 molecular-weight subunit, and its selenium was in the amino acid selenocysteine. The 75 000 molecular-weight protein was chromatographically distinct from glutathione peroxidase. In order to determine if these selenoproteins protect against cadmium toxicity, 109CdCl2 was administered to rats that were isotopically prelabeled with 75Se. At 3, 25 and 72 h after 109Cd administration, no 109Cd was associated with selenium-containing proteins. Two of the nonglutathione peroxidase selenoproteins were apparently unique to the kidney.     

Gel Filtration of Lignosulphonic Acids and Peptide-Precipitating Abilities of the Separated Fractions

Lignosulphonic acids in dialysed sulphite spent liquor and purified lignosulphonic acids were subjected to gel chromatography on Sephadex G-75, G-100 and G-200 and the fractions tested for peptide-precipitating ability. About 56 % of the total lignosulphonic acids in the dialysed sulphite spent liquor had estimated molecular weights above 90000 and about 72 % above 44000. About 94 % of the purified lignosulphonic acids had molecular weights above 90000 and the remaining 6 % had above 36000. The major peptide-precipitating activity of the lignosulphonic acids was due to fractions with molecular weights in excess of 90000. The percentage of peptides in the peptide-lignosulphonic acid precipitates was found to be 80–90. The molecular weights of the peptides used were found to have an upper limit of about 20000. The lower limit for molecular weights of lignosulphonic acid-precipitating peptides is estimated to be below 6000.     

Rat plasma selenoprotein P properties and purification

A selenoprotein in rat plasma, selenoprotein P, was fractionated and characterized. Plasma collected from rats 3 h post injection of 75SeO3(2-) contained one 75Se-labeled protein, selenoprotein P. Selenoprotein P was fractionated using salt precipitation, Affi-Gel Blue, and DEAE chromatography. The 75Se-containing subunit of selenoprotein P was purified to 90% homogeneity using SDS-polyacrylamide gel electrophoresis followed by electroelution. This isolation resulted in an 850-fold purification of the 75Se-containing subunit of selenoprotein P with a 15% yield of 75Se radioactivity. The molecular weight of selenoprotein P in plasma was 98,000. The 75Se-containing subunit of selenoprotein P had a molecular mass of 57 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing under nondenaturing conditions resulted in a band of 75Se radioactivity at pH 5.4. A comparison of Coomassie Blue- and silver-staining properties of selenoprotein P in SDS-polyacrylamide gels was made. Reverse-phase HPLC and Sephadex G-50 chromatography of tryptic peptides of the 57 kDa subunit of selenoprotein P yielded several peaks of 75Se radioactivity. These results indicate that 75Se is present in several locations within the 57 kDa subunit of selenoprotein P.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

Evidence for specific selenium target tissues and new biologically important selenoproteins

After in-vivo labeling with [75Se]selenite the Se-containing proteins present in rat tissues were investigated by means of SDS-polyacrylamide gel electrophoresis. Thirteen Se-containing proteins or protein subunits with relative molecular weights of 12,100, 15,600, 18,000, 19,700, 22,200, 23,700, 27,800, 33,300, 55,500, 59,900, 64,900, 70,100 and 75,400 were detected in the tissue homogenates. The protein with the molecular weight of 23,700 was the subunit of glutathione peroxidase, which is the only selenoprotein so far known to have biological functions in animals. Most of these proteins were found in all tissues investigated but one was only detected in the testes and the spermatozoa and one was present mainly in the thyroid. With inadequate selenium intake there was a priority supply of the element to the brain, the reproductive and the endocrine organs, and at a molecular level to Se-containing proteins other than glutathione peroxidase. The results suggest important biological functions of these selenoproteins, especially in the specific target tissues.     

Reaction of selenium with immunoglobulin molecules.

Radioactive selenite reacts with purified human and goat immunoglobulins at acidic and neutral pH. The antigenic properties of the immunoglobulins are retained during the selenium labelling as shown by immunoelectrophoresis and autoradiography. Pepsin digests of 75Se-labelled IgG possess 75Se both in the (Fab')2 fraction and in the low molecular weight peptides derived from the Fc domains. Alpha-1-acid glycoprotein, ribonuclease, and lysozyme are also labelled by this procedure. Enhancement of 75Se incorporation by urea, guanidinium chloride, mercaptoethanol, sodium sulfite and carrier selenite is interpreted as an effect of destabilization of IgG disulfide bonds. Up to 1.4 g atoms Se per mol IgG have been incorporated. We assume that selenite is cleaving disulfides by a process akin to sulfitolysis. The lability of the isolated 75Se-labelled IgG to high concentrations of mercaptans and sulfite is consistent with this idea. These 75Se-labelled proteins may be useful in structure studies and radioimmunoassay.     

Selenoproteins from rat testis cytosol

Radioactive inorganic selenium, administered intraperitoneally at 1 mg/kg body weight to young adult rats, acculumulates in testes for 7 days or longer, whereas liver, kidney and serum levels fall more rapidly. 3–4 h after administration of [⁷⁵Se]selenite, 55–60% of the radioactivity in the testes was found in the cytosol, associated with protein. Ultragel ACA-22 chromatography of testis cytosol prepared 4 h after ⁷⁵Se treatment revealed a major selenoprotein having an apparent molecular weight of 59 000. Sodium dedecyl sulfate polyacrylamide gel electrophoresis indicated extensive heterogeneity of radioactivity with apparent molecular weights of about 57 000 and 45 000 and 15 000. Cytosol from rats treated 4 weeks earlier showed predominance of the 15 000 molecular weight [⁷⁵Se]selenoprotein. Sucrose density gradient ultracentrifugation at either low or high ionic strength demonstrated a single 7 S selenoprotein. Chromatography with Blue-Sepharose indicated that the radioactivity was not associated with albumin. Strong ⁷⁵Se binding to protein was demonstrated by overnight dialysis against water, 2 M NaCl, β-mercaptoethanol, 8 M urea, selenite. However, 85% of the ⁷⁵Se was removed by dialysis against 0.5 M NaOh. This stability contrasts with the lability of disulfide reagents of selenite-protein complexes formed in vitro. The fact that selenium is incorporated in substantial amounts into a discrete and stable protein suggests a physiological role for this essential trace element in the testes.     

Subcellular distribution of selenoproteins in the liver of the rat

After in vivo labeling with [75Se]selenite, the intracellular distribution of selenoproteins in the liver was investigated in selenium-adequate and selenium-deficient rats. In the subcellular fractions, which were obtained by differential centrifugation, the proteins were separated by means of SDS-PAGE and the selenium compounds were identified via their 75Se activity. In this way twelve selenium-containing proteins or protein subunits with molecular weights between 12,100 and 75,400 were found. Glutathione peroxidase was concentrated in the cytosol and in the mitochondria. With the newly detected selenoproteins, some were enriched in the cytosol, one was mainly found in the nuclear fraction and some, which were present mainly in the mitochondrial and microsomal fractions, are most probably membrane-bound. In the liver of selenium-depleted rats the selenium administered was used predominantly to restore the levels of some of the newly found selenoproteins, while in the liver of selenium-adequate animals most of the selenium retained was incorporated into the glutathione peroxidase. The differences in the distribution among the subcellular fractions and the specific incorporation of the element in selenium deficiency into certain compounds suggest that there are several metabolic pathways for selenium and that the selenoproteins are involved in several different processes of intracellular metabolism.     

Multiple selenocysteine content of selenoprotein P in rats

Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport.     

Some characteristics of 75Se-P,a selenoprotein found in rat liver and plasma,and comparison of it with selenoglutathione peroxidase

The selenoenzyme glutathione peroxidase cannot account for all the physiological effects of selenium in rat liver. Therefore, a study was carried out with the ultimate aim of identifying selenoproteins other than glutathione peroxidase. The incorporation of ⁷⁵Se, given as ⁷⁵SeO₃^2?, into centrifugally separated fractions of selenium-deficient and control rat livers was determined. In selenium-deficient liver much less ⁷⁵Se was incorporated into the 105,000g supernatant fraction than in controls, so this fraction was studied further by gel filtration, ion-exchange, and hydroxylapatite chromatography. Selenoglutathione peroxidase and another selenoprotein, called ⁷⁵Se-P, were separated and identified. Both these selenoproteins were also found in plasma. Selenium deficiency had opposite effects on incorporation of ⁷⁵Se by these proteins. It decreased ⁷⁵Se incorporation by glutathione peroxidase at 3 and 72 h after ⁷⁵Se injection but increased ⁷⁵Se incorporation by ⁷⁵Se-P. This suggests that ⁷⁵Se-P competes for available selenium better than does glutathione peroxidase when the element is in short supply. Apparent molecular weights of ⁷⁵Se-P from liver and plasma determined by gel filtration were, respectively, 83,000 and 79,000, which indicate proteins smaller than glutathione peroxidase. Cycloheximide pretreatment of the rat blocked ⁷⁵Se incorporation into plasma ⁷⁵Se-P. These experiments establish the existence of a selenoprotein, ⁷⁵Se-P, in rat liver and plasma which is chromatographically distinct from glutathione peroxidase and which incorporates ⁷⁵Se differently from glutathione peroxidase. ⁷⁵Se-P may account for some of the physiological effects of selenium.     

Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.     

SELENIUM AND GLUTATHIONE PEROXIDASE IN DEVELOPING RAT BRAIN1

Abstract– Week-old rats were given a subcutaneous injection of carrier-free Na₂⁷⁵SeO₃ and brain ⁷⁵Se distribution was studied after 30 days, with special reference to the selenoprotein, glutathione peroxidase (GSH-Px). Chemical fractionation studies showed the ⁷⁵Se was associated mainly with protein and not extracted by hot trichloroacetic acid or chloroform-methanol. Subcellular fractions also revealed a parallel distribution of ⁷⁵Se and protein with the notable exception that ⁷⁵Se was concentrated in the mitochondria and reduced in the cytosol. GSH-Px activity was demonstrated in the isolated mitochondrial fraction. The estimated biological half-life of brain ⁷⁵Se was 45 days. Gel filtration (Sephadex G-150) of brain cytosol resulted in four ⁷⁵Se peaks: peak 1 was associated with the void volume, and had the greatest ⁷⁵Se content; peak 2 (V_e/V_o= 1.4) contained nearly as much ⁷⁵Se and had an apparent molecular weight of 94,000; peak 3 (V_e/V₀= 2.4) had an apparent molecular weight of 13,500 and was markedly increased when brain was homogenized in the presence of Triton X-100; peak 4 consisted of low molecular weight compounds. When fresh cytosol (with or without Triton X-100) was chromatographed on Sephadex G-150, GSH-Px was detectable only in the void volume; however, storage of cytosol prepared in the presence of Triton X-100 shifted most of the activity to peak 2 (94,000). The GSH-Px activity in the void volume resembled the purified enzyme with regard to pH dependence, K_m for cumene hydroperoxide at fixed [GSH], and first-order kinetic behavior with respect to GSH. A minor peak of GSH-Px activity showing zero-order kinetics with respect to GSH concentration and an apparent molecular weight of 46,000 was detected when larger amounts of protein were chromatographed. The concentration of rat brain Se measured by chemical analysis reached adult levels by 2 weeks after birth, an age when the level of GSH-Px had just begun to rise. It was estimated that only 1/5 of the total brain Se may be accounted for by its presence in GSH-Px, suggesting that the function of the majority of brain Se remains to be determined.     

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.     

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.     

Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.     

Products of the reaction of selenite with intracellular sulfhydryl compounds

The usual first step in the intracellular metabolism of exogenous selenite is its chemical reaction with glutathione to form selenodiglutathione (1). We have investigated whether selenite also reacts intracellularly with other SH compounds. HeLa cells were exposed to [75Se]selenite and lysed with SDS. Cellular proteins and nucleic acids were precipitated with trichloroacetic acid, and the acid-soluble fraction was analyzed by ion-exchange thin-layer chromatography (ion-exchange TLC) and autoradiography. In control cells, the major [75Se]-containing species detected can be identified by its mobility as selenodiglutathione. Two other species were detected, which can be identified as selenodimercaptoethylamine and the mixed selenotrisulfide of mercaptoethylamine and glutathione. In contrast, in cells that were depleted of glutathione (by treatment with buthionine sulfoximine), very little, if any, selenodiglutathione was detected. However, new [75Se]-containing species were detected, which can be identified as selenodicysteine and the mixed selenotrisulfide of cysteine and glutathione. The same species were detected when [75Se]selenite was added to the acid-soluble fraction of a cell extract (as opposed to living cells), confirming that these compounds can be formed by nonenzymatic reactions.     

Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.     

An external-sample liquid scintillation counting for 75Se and its application to selenoprotein detection

An external-sample liquid scintillation (LS) counting for the gamma emitter ⁷⁵Se has been developed. An expressly designed well-type LS vial and a 2,5-diphenyoxazole-1,4-bis(5-phenyl-2-oxazoyl)-benzene-xylene solution containing 35% tertrabutylzinn allow ⁷⁵Se to be counted in a standard LS counter with counting efficiency up to 43.2%, much higher than that of conventional LS counting method. This external sample LS has a good count rate linearity and exhibits low background count rates. After in vivo labeling with [⁷⁵Se]selenite, ⁷⁵Se distributions and the Se-containing proteins present in tissues of male rat were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, external-sample LS and γ-detector. Eight Se-containing proteins or protein subunits were detected to be Se-containing proteins or protein subunits in arterial wall, and their apparent molecular masses (Mr) were 76.4, 67.0, 57.4, 30.3, 25.4, 22.7, 21.7, and 15.1 kDa, respectively. In addition, eight ⁷⁵Se-labeled proteins (Mr: 66.8, 57.0, 43.1, 30.0, 24.8, 19.8, 18.0, and 14.8 kDa) were found in brain homogenates, and nine ⁷⁵Se-labeled proteins (Mr: 117.0, 78.0, 66.6, 57.2, 43.0, 38.1, 25.0, 20.1, and 18.0 kDa) were detected in testis homogenates. Some of them should be new biologically important selenoproteins that have not been identified so far.     

Structure and properties of human interferon-alpha from Namalwa lymphoblastoid cells.

The chromatographic properties of human interferon-alpha from Namalwa lymphoblastoid cells on Sephadex G-75 are described. The interferons are separated into two groups of four, with apparent molecular weights 19050 and 22000. Some of the latter form dimers at high concentrations. Fractions containing interferon were studied by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Seven of the components had apparent molecular weights in this system, after reduction, of between 18400 and 20900: one component is probably glycosylated and has an apparent molecular weight of 27500. Amino acid sequences of peptides derived from interferon mixtures were determined and are related to published sequences deduced from the nucleotide sequences of cloned complementary DNA coding for interferon-alpha. The results show that the major interferon-alpha species isolated from Namalwa cells do not undergo C-terminal processing. Amino acid analyses of peptides are presented in Supplementary Publication SUP 50117 (28 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.