Purification and characterization of a novel abundant protein in rat bile that binds azo dye metabolites and copper

Following intravenous administration to rats of the azo dye hepatocarcinogen 3'-methyl-N,N-dimethyl-4-aminoazo-[14C]benzene, 60-70% of the injected dose was recovered in bile in 2 h. Approximately 10% of bile radioactivity was trichloroacetic acid-precipitable, not extracted by n-butanol and non-dialyzable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bile followed by fluorography revealed two major and several minor proteins to which radiolabelled azo dye metabolites were bound; one of these major proteins (50 kDa) was purified from bile and shown to be homogeneous by SDS-polyacrylamide gel electrophoresis, isoelectric focusing (pI 7) under denaturing conditions and N-terminal analysis. The protein (MBP) comprises 15% of the total bile protein. Amino acid analysis revealed a preponderance of acidic and hydrophobic amino acids. The absorption spectrum of the native protein had a major peak at 280 nm and minor peaks at 345, 400, 600 and 650 nm. The fluorescence spectrum showed a major excitation maxima at 285 and 350 nm and corresponding emission maxima at 345 and 440 nm. Atomic absorption spectroscopy revealed 5 atoms of Cu per mol protein. Approximately 90% of the Cu was EPR silent. MBP did not react with antisera directed against rat serum IgA, albumin or ceruloplasmin; nor did these proteins react against antisera to MBP. Seven distinct peptide bands ranging from 5 to 18 kDa were obtained when MBP was subjected to CNBr cleavage and the digests were analyzed by SDS-polyacrylamide gel electrophoresis. The [14C]-Me-DAB derived radioactivity was present in only two of the peptides, indicating specific binding sites for azodye metabolites.     

The molecular weight of NADPH-cytochrome C reductase isolated by immunoprecipitation from detergent-solubilized rat liver microsomes

The molecular weight of detergent-solubilized NADPH-cytochrome c reductase from rat liver microsomes has been estimated to be 79,000. The method used for this determination involves immunoprecipitation of deoxycholate-solubilized enzyme from ¹²⁵I-labeled microsomal proteins. The antibody was prepared against a purified preparation of Bromelain-solubilized enzyme (molecular weight 71,000). The immunoprecipitate was then subjected to SDS-polyacrylamide gel electrophoresis and the enzyme located by ¹²⁵I-gamma counting.     

Stopped-Flow Circular Dichroism Study on Conformational Change of Alpha-Chymotrypsin by Sodium Dodecyl Sulfate

Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [³⁵S]methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispersed cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells.     

Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells

32P-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32P]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase-2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.     

Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate β-mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A⁺)-RNA. Poly (A⁺)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A⁺)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.     

Use of purified antipeptide sera of selected specificity for the detection of the simian virus 40 large T antigen by western blotting

Western blotting was used as a powerful alternative to immunoprecipitation for the detection of the simian virus 40 (SV40) large tumor (T) antigen. After resolution by electrophoresis on a SDS-polyacrylamide gel of a [¹⁵S]methionine labeled crude extract from SV40 infected monkey kidney cells, the separated proteins were transferred electrophoretically on nitrocellulose paper. T antigen was detected on nitrocellulose strips by using for the first time, specific, purified antipeptide monoclonal antibodies directed against the N- and C-terminal portions of the molecule, and¹²⁵I-labeled Protein A.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Fatty acylation of human platelet proteins: evidence for myristoylation of a 50 kDa peptide

Fatty acid acylation of platelet proteins was studied by measuring incorporation of [3H]palmitate and [3H]myristate after incubation at 37 degrees C for 4 h. About ten major radiolabeled proteins were detected after SDS-polyacrylamide gel electrophoresis and fluorography, for both fatty acids. Cleavage by hydroxylamine treatment indicated an ester bond of either palmitate or myristate to these proteins. Nevertheless, a single 50 kDa peptide was specifically modified by an amide-linked myristate. The functions of acylated proteins in platelets are still unknown, but their relation with DLPC-induced shape changes and vesicle shedding is excluded.     

Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein.

Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed.     

Biosynthesis and maturation of alpha-N-acetylglucosaminidase in normal and Sanfilippo B-fibroblasts

The biosynthesis of alpha-N-acetylglucosaminidase in normal and Sanfilippo B fibroblasts was studied by labeling cells with [35S]methionine and isolation of the enzyme by immunoprecipitation. The immunoprecipitated polypeptides were separated by polyacrylamide gel electrophoresis and visualized by fluorography. alpha-N-acetylglucosaminidase is synthesized as a precursor of an apparent mol. wt. of 87,000. Intracellular processing of the precursor yields two polypeptides of apparent mol. wts. of 73,000 and 76,000 via several intermediates. It is accomplished within 3 days after synthesis. Less than 30% of the newly synthesized precursor is secreted. In the presence of 10 mM NH4Cl, secretion is enhanced to more than 80%. In our study, no alpha-N-acetylglucosaminidase polypeptides could be detected in fibroblasts from patients affected with either the severe or mild form of Sanfilippo disease, type B.     

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1 microM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin-stimulated phospholipid methyltransferase phosphorylation was time- and dose-dependent. 32P-labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P-labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.     

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.     

Cell-free synthesis and processing of a large precursor of glutamate dehydrogenase of rat liver

The mitochondrial matrix protein glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using mRNA from free or membrane-bound polysomes from rat liver. Immunoprecipitation of the (³⁵S)methionine labeled translation mixture was performed using rabbit anti-glutamate dehydrogenase serum. Analysis after electrophoresis of the immunoprecipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed that the glutamate dehydrogenase is synthesized ‘in vitro’ as a large precursor. A mitochondrial extract from rat liver processed the precursor synthesized “in vitro” to the mature form.     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Monoclonal antibodies that distinguish between human aorta smooth muscle and endothelial cells

A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor immunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

Extraction of histones H2A, H3 and H4 from yeast nuclei. Measurement of the extent of yeast histone acetylation following one-dimensional gel electrophoresis

Yeast histones H2A, H3 and H4 were specifically extracted from purified nuclei using a 2% NaCl/75% ethanol solution. The extraction resulted in the complete removal of H2A, H3 and H4 from the nuclear pellet, as monitored by SDS-polyacrylamide gel electrophoresis of the protein. The relative absence of nonhistone proteins from this histone subset simplifies the determination of the extent of histone modification in yeast. Levels of H4 acetylation were measured directly on Coomassie blue-stained Triton acid-urea gels and the levels verified by gel fluorography of the [3H]acetate-labeled histone.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.     

A protein-tyrosine kinase in the nuclear matrix from rat liver

Protein kinase activity in isolated nuclei from rat liver was detected in situ after electrophoresis on SDS-polyacrylamide gel containing no exogenous protein substrate. After renaturation of polypeptides, the gel was incubated with [gamma-32P]ATP and divalent cations. Among five major protein kinase activities observed as radioactive bands by autoradiography, a protein kinase autophosphorylating on tyrosine (Mr 30,000) was identified and found to be localized in the nucleus, particularly in the nuclear matrix. The intensity of the activity band representing the level of the protein-tyrosine kinase in rat liver nuclei did not appreciably change during 3-24 h after partial hepatectomy.