Subcellular distribution of selenium and Se-containing proteins in human liver

Selenium is an essential trace element in many living organisms. In the present paper, the subcellular distribution of selenium and Se-containing proteins in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation and column chromatography. Selenium was mainly enriched in nuclei, mitochondria and cytosol. Almost half of Se existed in the nuclei due to their large amount in liver and high Se concentration. 15-30% of Se was found in small compounds with Mr<2000 in the liver components separated by dialysis. The average abundance of Se in small molecular mass species of whole-liver was 23.6%, which suggested most of Se associated with biological macromolecules. Eight kinds of Se-containing proteins with molecular mass of 335+/-20, 249+/-15, 106+/-11, 84.6+/-5.8, 70. 5+/-5.4, 45.6+/-1.5, 14.8+/-2.6, 8.5+/-1.2 kDa were found in the subcellular fractions of human liver. Among them the 335, 84.6 and 8. 5 kDa proteins were individually present in one subcellular fraction, whereas the others coexisted in two, three or four subcellular fractions. The most abundant Se-containing proteins, 70.5 and 14.8 kDa, accounted for 33.6% and 48.5% in the whole-liver soluble Se-containing protein, respectively. The former was enriched in cytosol and the latter was mainly present in nuclei and mitochondria.     

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.     

Molecular weights and hydrophobicity of the polypeptide chain of sarcoplasmic reticulum calcium(II) adenosine triphosphatase and of its primary tryptic fragments.

The polypeptide chain of the Ca2+-stimulated adenosine triphosphatase from sarcoplasmic reticulum has a molecular weight of 119 000+/-6500 on the basis of sedimentation equilibrium measurements in sodium dodecyl sulfate. The two primary fragments obtained by limited proteolysis each have within experimental error the same molecular weight, corresponding to one-half the molecular weight of the whole chain. Both fragments are eqaully resistant to complete denaturation by guanidine hydrochloride, a property characteristic of many intrinsic membrane proteins. This suggests that the native enzyme has two membrane-embedded halves, with an externally accessible link between them.     

Protein-protein interactions in contact activation of blood coagulation. Characterization of fluorescein-labeled human high molecular weight kininogen-light chain as a probe

Limited proteolysis of high molecular weight kininogen by kallikrein resulted in the generation of an inactive heavy chain of Mr = 64,000 and active light chains of Mr = 64,000 and 51,000 when analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions. Starting with kininogen from outdated plasma, a light chain with an apparent molecular weight of 51,000 on 7.5% SDS gels was purified and characterized. Molecular weights of 28,900 +/- 1,100 and 30,500 +/- 1,600 were obtained by gel filtration of the reduced and alkylated protein in 6 M guanidine HCl and equilibrium sedimentation under nondenaturing conditions in the air-driven ultracentrifuge, respectively. The light chain stained positively with periodic acid-Schiff reagent on SDS gels indicating that covalently attached carbohydrate may be responsible for the anomalously high molecular weight estimated by SDS-gel electrophoresis. A single light chain thiol group reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence and absence of 6 M guanidine HCl. Specific fluorescent labeling of the thiol group with 5-(iodoacetamido)fluorescein (IAF) occurred without loss of clotting activity. Addition of purified human plasma prekallikrein to the IAF-light chain resulted in a maximum increase in fluorescence anisotropy of 0.041 +/- 0.001 and no change in the fluorescence intensity. Fluorescence anisotropy measurements of the equilibrium binding of prekallikrein to the IAF-light chain yielded an average Kd of 17.3 +/- 2.5 nM and stoichiometry of 1.07 +/- 0.07 mol of prekallikrein/mol of IAF-light chain. Measurements of the interaction of prekallikrein with iodoacetamide-alkylated light chain using the IAF-light chain as a probe gave an average Kd of 16 +/- 4 nM and stoichiometry of 1.0 +/- 0.2 indicating indistinguishable affinities for prekallikrein.     

Molecular structure of aminopeptidase A from bovine kidney

Using gel-filtration through Sephadex G-100 and polyacrylamide gel electrophoresis in the presence of 0,5% sodium dodecyl sulfate, it was found that aminopeptidase A has a molecular weight of 65 000 +/- 2000 and is made up of two subunits with mol. weights of 33 000 +/- 2000. Each subunit consists of two polypeptide chains with mol. weights of 22 000 +/- 2000 and 12 000. During enzyme dissociation into subunits the aspartylnaphtylamidase activity is lost, while the glutamylnaphtylamidase activity is retained.     

Radiation inactivation of multispecific transport systems for bile acids and xenobiotics in basolateral rat liver plasma membrane vesicles.

The functional molecular mass of the cholate, phallotoxin, iodipamide, and ouabain transport proteins in isolated basolateral plasma membrane vesicles was determined by radiation inactivation. Purified basolateral plasma membrane vesicles were irradiated (-90 to -120 degrees C) with high energy electrons from a 10-MeV linear accelerator at doses from 0 to 30 megarads. After each dose, the initial uptake, the equilibrium binding, and the binding of the substrates at 4 degrees C were checked. The size of the transporting function was, for cholate, 107 +/- 8.9 kDa; for phallotoxin, 104 +/- 7 kDa; and for ouabain, 120 +/- 4.7 kDa. The target size for the binding proteins was 56 +/- 4.2, 57 +/- 5, and 47.2 +/- 1.95 kDa for cholate, phallotoxin, and taurocholate, respectively. In the case of iodipamide, the functional molecular mass for both the transport and binding proteins was 54 +/- 4.8 kDa.     

Proteome profiling for assessing diversity: analysis of individual heads of Drosophila melanogaster using LC-ion mobility-MS

The proteomes of three heads of individual Drosophila melanogaster organisms have been analyzed and compared by a combination of liquid chromatography, ion mobility spectrometry, and mass spectrometry approaches. In total, 197 proteins are identified among all three individuals (an average of 120 +/- 20 proteins per individual), of which at least 101 proteins are present in all three individuals. Within all three datasets, more than 25 000 molecular ions (an average of 9000 +/- 2000 per individual) corresponding to protonated precursor ions of individual peptides have been observed. A comparison of peaks among the datasets reveals that peaks corresponding to protonated peptides that are found in all heads are more intense than those features that appear between pairs of or within only one of the individuals. Moreover, there is little variability in the relative intensities of the peaks common among all individuals. It appears that it is the lower abundance components of the proteome that play the most significant role in determining unique features of individuals.     

Interactions of the Pseudomonas aeruginosa outer membrane proteins with plasma fibronectins. Bacterial adhesin investigation

Pseudomonas aeruginosa adherence is a complex phenomenon largely mediated by pili involving specific receptor-ligand interactions. Anti-fibronectin antibodies as well as plasmatic fibronectin are able to inhibit P. aeruginosa adherence onto A549 cells showing that matricial fibronectin is an actual receptor for this bacterium. Experiments performed in vitro with human plasmatic fibronectin used as receptor and outer membrane proteins of P. aeruginosa as ligands show the presence of four fibronectin-binding proteins. These proteins with molecular mass of 70 +/- 2, 60 +/- 2, 48 +/- 2 and 36 +/- 1 kDa should be adhesins of P. aeruginosa on epithelial cell matrix in a non-pilus mediated adherence.     

Studies on aconitase species from Saccharomyces cerevisiae, porcine and bovine heart, obtained by a modified isolation method

Aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) was isolated from Saccharomyces cerevisiae, porcine and bovine heart by a simplified method including affinity chromatography on Blue Dextran-Sepharose. Partial characterisation reveals that the aconitase species are all similar due to molecule size, amino acid composition, isoelectric point and enzymatic activity. Aconitase appears as a single polypeptide chain with a small carbohydrate content. A molecular weight of 79000 +/- 2000 and a Svedberg constant of s20,w = 4.75 +/- 0.2 S indicate a compact structure of aconitase. Due to different properties among the yeast aconitase species concerning isoelectric point and enzymatic activity a coherence between net charge of the protein and redox state of the Fe-S cluster can be expected.     

The composition of serval (Felis serval) milk during mid-lactation

Milk was obtained from three captive servals. The average nutrient content was 158.3+/-44.4 g protein; 152.6+/-62.3 g fat; and 68.7+/-31.4 g lactose per kg milk. The protein fraction respectively consisted of 117.7+/-44.8 g caseins per kg milk and of 40.6+/-6.7 g whey proteins per kg milk. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cheetah and cat milk, with small differences in the beta-caseins. The lipid fraction contains 313.3+/-18.8 g saturated and 338.6+/-11.9 g mono unsaturated fatty acids per kg milk fat respectively. The high content of 292.4+/-24.9 g kg(-1) milk fat of polyunsaturated fatty acids is due to a high content in linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed. In general, serval milk has a higher protein and fat content than that of the domestic cat and cheetah, and a lower content of unsaturated fatty acids than that of the domestic cat.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

D-Glucose-sensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter

Cytochalasin B was found to bind to at least two distinct sites in human placental microvillous plasma membrane vesicles, one of which is likely to be intimately associated with the glucose transporter. These sites were distinguished by the specificity of agents able to displace bound cytochalasin B. [3H]Cytochalasin B was displaceable at one site by D-glucose but not by dihydrocytochalasin B; it was displaceable from the other by dihydrocytochalasin B but not by D-glucose. Some binding which could not be displaced by D-glucose + cytochalasin B binding site. Cytochalasin B can be photoincorporated into specific binding proteins by ultraviolet irradiation. D-Glucose specifically prevented such photoaffinity labeling of a microvillous protein component(s) of Mr = 60,000 +/- 2000 as determined by urea-sodium dodecyl sulfate acrylamide gel electrophoresis. This D-glucose-sensitive cytochalasin B binding site of the placenta is likely to be either the glucose transporter or be intimately associated with it. The molecular weight of the placental glucose transporter agrees well with the most widely accepted molecular weight for the human erythrocyte glucose transporter. Dihydrocytochalasin B prevented the photoincorporation of [3H]cytochalasin B into a polypeptide(s) of Mr = 53,000 +/- 2000. This component is probably not associated with placental glucose transport. This report presents the first identification of a sodium-independent glucose transporter from a normal human tissue other than the erythrocyte. It also presents the first molecular weight identification of a human glucose-insensitive high-affinity cytochalasin B binding protein.     

Pallidin. Purification and characterization of a carbohydrate-binding protein from Polysphondylium pallidum implicated in intercellular adhesion.

A carbohydrate-binding protein from Polysphondylium pallidum, a species of cellular slime mold, was purified to homogeneity by adsorption to formalinized erythrocytes and elution with D-galactose. The protein, for which we propose the name PALLIDIN, is assayed by its activity as an agglutinin of erythrocytes. It was previously shown to have different carbohydrate-binding specificities than discoidin, a carbohydrate-binding protein from Dictyostelium discoideum, another species of slime mold. Evidence has been presented previously that each of these proteins is detectable on the cell surface. In the present report we show that the physico-chemical properties of pallidin are different from discoidin. Pallidin has a subunit molecular weight of 24 800 +/- 1100 determined by polyacrylamide electrophoresis in the presence of dodecyl sulfate and 2-mercaptoethanol, compared to 26 100 +/- 1000 for discoidin. The weight-average molecular weight of pallidin is 250 000 +/- 50 000 determined by equilibrium sedimentation in the presence of D-galactose compared to 100 000 +/- 2000 for discoidin. In equilibrium sedimentation studies, pallidin exhibited some heterogeneity at equilibrium while discoidin was homogeneous. The amino acid composition of pallidin is generally similar but clearly different from the composition of discoidin. The isoelectric point of pallidin is 7.0 compared to 6.1 for discoidin. Like discoidin, pallidin contains no detectable hexosamine or neutral sugar. These results establish that agglutinins from two species of cellular slime molds are distinct. The different properties of the cell-surface agglutinins, pallidin and discoidin, are consistent with their suggested role in species-specific cellular recognition and adhesion in the species of slime mold from which they are derived.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

Shape of proteins S3 and S17 from the small subunit of Escherichia coli ribosome.

The shapes of proteins S3 and S17 purified from the 30 S subunit of Escherichia coli A19 were studied by hydrodynamic methods. The proteins have s020,w values of 2.1 +/- 0.1 S and 1.2 +/- 0.1 S and D020,w values of 7.4 +/- 0.5 . 10(-7) cm2/s and 11.4 +/- 0.6 . 10(-7) cm2/s. The respective molecular weights determined by sedimentation equilibrium are 25 800 +/- 500 and 9900 +/- 300. The intrinsic viscosity values for the two proteins are 5.8 +/- 0.3 ml/g and 4.2 +/- 0.2 ml/g. From these hydrodynamic parameters a slightly elongated shape for S3 and a globular shape for S17 have been concluded.     

Hyperoxia and recovery from hypoxia alter collagen in peripheral pulmonary arteries similarly

Chronic hypoxia causes pulmonary hypertension, the mechanism of which includes altered collagen metabolism in the pulmonary vascular wall. This chronic hypoxic pulmonary hypertension is gradually reversible upon reoxygenation. The return to air after the adjustment to chronic hypoxia resembles in some aspects a hyperoxic stimulus and we hypothesize that the changes of extracellular matrix proteins in peripheral pulmonary arteries may be similar. Therefore, we studied the exposure to moderate chronic hyperoxia (FiO2 = 0.35, 3 weeks) in rats and compared its effects on the rat pulmonary vasculature to the effects of recovery (3 weeks) from chronic hypoxia (FiO2 = 0.1, 3 weeks). Chronically hypoxic rats had pulmonary hypertension (Pap = 26 +/- 3 mm Hg, controls 16 +/- 1 mm Hg) and right ventricular hypertrophy. Pulmonary arterial blood pressure and right ventricle weight normalized after 3 weeks of recovery in air (Pap = 19 +/- 1 mm Hg). The rats exposed to moderate chronic hyperoxia also did not have pulmonary hypertension (Pap = 18 +/- 1 mm Hg, controls 17 +/- 1 mm Hg). Collagenous proteins isolated from the peripheral pulmonary arteries (100-300 microm) were studied using polyacrylamide gel electrophoresis. A dominant low molecular weight peptide (approx. 76 kD) was found in hypoxic rats. The proportion of this peptide decreases significantly in the course of recovery in air. In addition, another larger peptide doublet was found in rats recovering from chronic hypoxia. It was localized in polyacrylamide gels close to the zone of alpha2 chain of collagen type I. It was bound to anticollagen type I antibodies. An identically localized peptide was found in rats exposed to moderate chronic hyperoxia. The apparent molecular weight of this collagen fraction suggests that it is a product of collagen type I cleavage by a rodent-type interstitial collagenase (MMP-13). We conclude that chronic moderate hyperoxia and recovery from chronic hypoxia have a similar effect on collagenous proteins of the peripheral pulmonary arterial wall.     

Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins.

1. A large-scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non-haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 +/- 300 nmol of acetylene reduced/mg protein per min in the presence of sautrating amounts of the other. This procedure freed the Mo-Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94. 2. Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis. Both proteins were oxygen-sensitive but not cold-labile. Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml. 3. The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 +/- 0.3 atoms of Mo, 23 +/- 2 atoms of Fe, 20 +/- 2 acid-labile sulphide and 47 tryptophan residues/mol. The protein consists of 4 subunits of mol. wt 60 000 (approx.). The reduced protein showed electron paramagmetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6. Upon oxidation abosrbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm. 4. The smaller protein had a molecular weight of 64 000 and contained 4 g-atoms of Fe and 4 acid-labile sulphide groups/mol but no tryptophan. It had two subunits of mol. wt 30 800. The reduced protein showed electron paramagnetic resonance signhe protein retained almost full activity after oxidation with phenazine methosulphate. The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen-inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm. Oxygen-inactivated protein showed a sharp peak at 277.5 nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm. 5. Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues. Analyses of compositional relatedness showed that the nitrogenase proteins from A. chroococcum were most closely related to those from A. vinelandii and least so to those from Clostridium pasteurianum.