Gap junctions from the lens: Purification and characterization by chemical crosslinking reagent

Quantitation of gap junction preparations from chick lens by transmission electron microscopy has indicated that 95.0% of the membrane bilayer material is in the form trilayer structures. The preparations were comprised of a major polypeptide component of 26K, as well as minor components of 49K, 46K and 22K–15K. Treatment with oxidizing agent resulted in the production of apparent homo-oligomeric complexes involving the 26K and 46K components. These results demonstrate that the 26K polypeptide is the major component of highly purified preparations of lens gap junctions. Furthermore, they demonstrate that this 26K component plus an additional 46K component are both involved in extensive nearest-neighbor interactions in the intact junctional complex.     

Lens calcium activated proteinase: Degradation of vimentin

The lens has been shown to contain a Ca⁺² activated proteinase specific for vimentin. The proteinase is present in the soluble fraction of the cortex but not in the epithelium. It is suggested that this proteinase is expressed during terminal differentiation of the epithelial cells and may be responsible for degradation of the intermediate filaments in the fiber cells. The proteinase is inhibited by EGTA but not by several proteinase inhibitors.     

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

Macromolecular covalent binding of [14C]nitrobenzene in the erythrocyte and spleen of rats and mice

Nitrobenzene exposure is known to produce red blood cell damage as well as engorgement and sinusoidal congestion of the spleen in male Fischer-344 (F-344) rats but not in male B6C3F1 mice. These studies were conducted to investigate the species differences in the covalent binding of [¹⁴C]nitrobenzene in the erythrocyte and spleen and to assess the contribution of nitrobenzene-induced erythrocytic damage to the splenic effects. Total and covalently bound ¹⁴C concentrations in erythrocytes of rats were 6–13 times greater than those of mice following a single oral dose of 75, 150, 200 or 300 mg/kg [¹⁴C]nitrobenzene, suggesting that species differences in nitrobenzene-induced red blood cell toxicity may be related to differences in erythrocytic accumulation of nitrobenzene and its metabolites. Covalently bound ¹⁴C in erythrocytes of rats peaked 24 h following administration of 200 mg [¹⁴C]nitrobenzene/kg; in contrast, bound radio-label in erythrocytes from mice plateaued at 10 h. Splenic engorgement increased in a time-related manner in treated rats but not in mice. Species specificity was also observed in the accumulation of bound radiolabel in the spleen. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lysed, dialyzed erythrocytes from treated rats revealed that hemoglobin was the primary, if not the exclusive, site of macromolecular covalent binding following nitrobenzene treatment. SDS-PAGE of dialyzed rat spleens revealed that 82% of total bound ¹⁴C migrated identically to hemoglobin. These data indicate that covalent binding of [¹⁴C]nitrobenzene and its metabolites in the spleen is primarily derived from bound ¹⁴C from scavenged erythrocytes. Therefore, the species differences in splenic engorgement and accumulation of [¹⁴C]nitrobenzene may be related to differences in susceptibility to nitrobenzene-induced red blood cell damage.     

Evidence for the occurrence of calmodulin in the yeasts Candida albicans and Saccharomyces cerevisiae

The lectin extracted from Vicia graminea seeds has been purified by conventional techniques but such procedures did not give a satisfactory yield. We describe a new purification which involves 3 steps after obtention of the crude extract. The first step is based on affinity chromatography on con A—Sepharose. Further purification steps were performed on DEAE-Sephacel chromatography and ultrogel AcA₄₄ gel filtration. The homogeneity of the lectin was demonstrated by polyacrylamide gel electrophoresis. Purification of the lectin by this new method was less time consuming, the yield was higher and the specific activity increased.     

Expression,purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2

Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280 kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12–320) and N2 (aa12–860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23 kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both α helixes and β sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.     

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used α- and ß-amylase and α- and ß-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of α-glucosidase and ß-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ß-glucosidase and α-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the α- and ß-glycosidases.     

A new protein of the brain myelin: isolation and chemical characterization

An unknown protein has been isolated from bovine brain myelin. This protein, purified in the nonionic detergent n-octylpolydisperse oligooxyethylene, reveals on SDS gel electrophoresis a large number of bands in the higher MW region. However, chemical analysis and gel chromatography indicate the presence of a single, small protein containing large amounts of bound phosphatidylserine. N-terminal and C-terminal sequences, aminoacid composition, and the anomalous electrophoretic behaviour led us to exclude the protein as a fragment of other already known myelin proteins.     

Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting "fibrillar" structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.     

Effects of common radioiodination procedures on the binding of glycoproteins to immobilized lectins

Representative glycoproteins including fetuin, protein A, ovalbumin, α₁ acid glycoprotein, and the major glycoprotein of equine infectious anemia virus were labelled with ¹²⁵I by the chloramine-T or Bolton-Hunter procedure and their binding to immobilized Con A or lentil lectin compared to untreated samples of each glycoprotein. Glycoprotein modification was no greater than one substituted residue per protein molecule. Yet the radioiodinated glycoproteins typically displayed only 0–50% of the lectin binding observed with untreated samples. These results indicate that lectin glycoprotein binding can be markedly altered by minor modifications in protein structure.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.     

Characterization of a zebrafish estrogen-sulfating cytosolic sulfotransferase: inhibitory effects and mechanism of action of phytoestrogens

Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.     

Detection of enzymatic activities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerases as model enzymes

Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations. Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here. It was observed that reproducibility of a reported procedure for DNA polymerase detection (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucl. Acids Res.9, 1825–1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al. A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described. The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis. These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase I and its Klenow fragment and nanogram amounts of calf thymus α and rat liver (Novikoff hepatoma) β polymerases. Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities.     

A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids

A rapid method based on a defined methanol-chloroform-water mixture for the quantitative precipitation of soluble as well as hydrophobic proteins from dilute solutions (e.g., column chromatography effluents) has been developed. The effectiveness of this method is not affected by the presence of detergents, lipids, salt, buffers, and beta-mercaptoethanol.     

Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris

Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700 μg/ml). D-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50 L bioreactor at a 20 L working volume. After 48 h fermentation with continuous feeding of 25% (w/v) D-sorbitol and 0.8% PTM4, the cell grew to A(600)=178 and intracellularly expressed Canstatin-N reached 780 mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340).