Purification of BBF, a DNA-binding protein recognizing a positive cis-acting element in the mouse alpha 1(III) collagen promoter.

A positive cis-acting element, the B element, located between -83 and -61 in the mouse alpha 1(III) collagen promoter, binds a factor present in nuclear extracts of NIH 3T3 fibroblasts and HeLa cells. We have purified this factor using ion exchange chromatography, sequence-specific DNA affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel fractionation. The DNA sequence used for the affinity chromatography was a single-base substitution in the B element that increased the stability of the B element-protein complex by 50%. Purification of the B element-binding factor (BBF) by DNA affinity chromatography resulted in the apparent loss of most or all of the DNA-binding activity of this factor. The DNA-binding activity could, however, be reconstituted by combining two chromatographic fractions: the high-salt eluate and the column flow-through. When the partially purified high-salt eluate was size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent renaturation of gel fractions from guanidine HCl, the purified BBF (apparent molecular weight of about 95,000) bound to the B element with high affinity. These results suggest that during DNA affinity purification of BBF a factor that inhibits BBF DNA binding was co-eluted with BBF. This inhibition of BBF DNA binding was reversed by the addition of the DNA affinity column flow-through. The binding of BBF to the B element of the mouse alpha 1(III) collagen promoter is therefore an apparently complex process involving interactions between BBF and other protein factors.     

Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Purification and partial characterization of protein phosphatases from rat thymus

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.     

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.     

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.     

Activator protein supporting the botulinum ADP-ribosyltransferase reaction

The ADP-ribosyl moiety of NAD was transferred to proteins with Mr values of 22,000 and 25,000 when bovine brain cytosol was incubated with a botulinum ADP-ribosyltransferase C3 (BT-C3) which was purified from the culture medium of a type C strain of Clostridium botulinum. Any protein fraction eluted from a chromatographic column to which the cytosol had been applied, however, was not significantly ADP-ribosylated by BT-C3, unless the reaction mixture was further supplemented with a small amount of the cytosol. Thus, substrate protein(s) could be partially purified based on their ability to be ADP-ribosylated by BT-C3 in the presence of the cytoplasmic activator(s). The rate of ADP-ribosylation of the substrates was extremely low by itself but was increased enormously and progressively when increasing amounts of cytosol were added, affording a reliable means for assay of the activator contained therein. The activator was separated from the substrate proteins and partially purified from the cytosol by sequential chromatography steps with an anion exchanger and a gel filtration column. The activity of the partially purified activator was heat-labile and protease-sensitive, suggesting that the activator was a protein or had a protein component necessary for activity. The action of the activator protein(s) was specific for BT-C3-catalyzed ADP-ribosylation; cholera toxin-catalyzed ADP-ribosylation of GTP-binding protein (Gs) was not supported by this activator. Thus, this is the first report to show that botulinum ADP-ribosyltransferase-catalyzed reaction can proceed significantly only in the presence of other protein factor(s), just as has been observed with an ADP-ribosylation factor required for cholera toxin-induced similar reaction.     

Cytosol induces apparent selectivity of glucocorticoid receptor binding to nucleic acids of different secondary structure

Unpurified rat liver glucocorticoid-receptor complexes within cytosol show a distinct binding preference for double-stranded DNA over single-stranded DNA; the binding to Escherichia coli rRNA is negligible. Extensive purification of the receptor abolishes its ability to distinguish among DNAs of different secondary structure and the affinity of the purified receptor toward RNA is greatly enhanced, reaching 30–50% of that of DNA. The purification effect is reversible: after cytosol addition to purified receptor preparation the binding preference restores. NaCl does not mimic the effect of cytosol. The flow-through fraction of a phosphocellulose column retains the ability of crude cytosol to produce selective decrease in the receptor binding to single-stranded DNA. This effect may also be observed by using two types of DNA-cellulose bearing double-stranded or denatured DNA, pretreated with crude cytosol. Additionally, pretreatment of immobilized DNA with even low cytosol concentrations has been shown to markedly enhance receptor binding, although this enhancement was lacking specificity with respect to DNA secondary structure. The nature of cytosolic active principle and some possible regulatory implications are discussed.     

Maternal Exogastrula-Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE-cellulose, the exogastrula-inducing activity was found in the flow-through fraction at all developmental stages. In particular, the activity present in the flow-through fraction of unfertilized eggs represents the presence of maternal exogastrula-inducing peptides (EGIPs). The flow-through fractions from the column of DEAE-cellulose were applied to a column of Sephadex G-100 and the activities in the eluate were assayed. The active low-molecular-weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP-D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.     

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.     

Intracellular apoptosis-inducing factor is induced by a vacuolar type H+-ATPase inhibitor in B lineage cells

We previously reported that concanamycin A, a specific inhibitor of vacuolar type H+-ATPases, induces DNA fragmentation in B cell hybridoma HS-72 cells. In the present study, we found that the cytosol from concanamycin A-treated HS-72 cells had a cytotoxic effect on intact cells in a cell viability assay. While activin A also induced apoptosis in HS-72 cells, the cytosol from activin A-treated HS-72 cells had no effect on cell viability. We purified the cytosol from concanamycin A-treated HS-72 cells by a four-step procedure: ultracentrifugation; HiTrap heparin column chromatography; HiTrap Q column chromatography; and reverse-phase high performance liquid chromatography on a C18 hydrophobic support. The biologically active fraction, which was used as partially purified cytosol, gave a specific band of protein with a molecular mass of 33 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis in cells cultured with the partially purified cytosol. The overexpression of human Bcl-2 suppressed apoptosis, indicating that the cytosol from concanamycin A-treated HS-72 cells induces apoptosis by a Bcl-2-inhibiting mechanism. These findings suggest that concanamycin A, a vacuolar type H+-ATPase inhibitor, produces intracellular apoptosis-inducing factor in B cell hybridoma.     

Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose.

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.     

Characteristics of solubilized human-somatotropin-binding protein from the liver of pregnant rabbits.

A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 x 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.     

Affinity purification of insulin-degrading enzyme and its endogenous inhibitor from rat liver.

A metallothiol protease called insulin-degrading enzyme (IDE) seems to be implicated in insulin metabolism to terminate the response of cells to hormone, as well as in other biological functions, including muscle differentiation, regulation of growth factor levels, and antigen processing. In order to obtain highly pure and biologically active IDE, we have developed an immunoaffinity method using a monoclonal antibody to this enzyme (9B12). When the cytosolic fraction of rat liver was first applied to a 9B12-coupled Affi-Gel 10 column, more than 97% of the insulin-degrading activity was absorbed. Among various kinds of buffers successfully eluting the enzyme, only the buffer with a high pH (pH 11) could retain the full biological activity of this enzyme. IDE was further purified via two steps of chromatography using Mono Q anion exchange and Superose 12 molecular sieve columns. The final preparation showed a single band at 110 kDa on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the eluate from the immunoaffinity column, the inhibitory activity associated with the enzyme was also observed. To better recover this endogenous inhibitor, heat-treated cytosolic fraction was fractionated by ammonium sulfate precipitation and applied to the immunoaffinity column on which IDE had been adsorbed. Then, IDE and its inhibitor could be co-eluted with pH 11 as a complex form. After heat treatment of this fraction, the inhibitor was further purified using the same series of chromatography as IDE to more than 20,000-fold; it showed a 14 kDa band on SDS-PAGE. It inhibited both the insulin degradation by IDE in a competitive manner and the cross-linking of 125I-insulin to IDE. Highly purified IDE and the endogenous inhibitor will be useful tools for better understanding the various biological functions of this enzyme.     

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).     

Affinity purification of human brain tissue factor utilizing factor VII bound to immobilized anti-factor VII

An efficient procedure for affinity purification of human tissue factor apoprotein that requires binding of only microgram quantities of human factor VII to anti-factor VII agarose is described. Factor VII was added to a 2% Triton X-100 extract of acetone brain powder in the presence of calcium. The resultant factor VII/tissue factor/calcium complex was bound to anti-factor VII-agarose, and the bound tissue factor was then eluted with EDTA. The eluate was passed through anti-goat IgG-agarose to remove contaminating goat IgG that leaks from the anti-factor VII column. Yield (units of activity) was 27%; specific activity was 2400 U/mg, which corresponds to that reported by others. The purified apoprotein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 47,000. Immunostaining with a goat anti-tissue factor IgG raised against the purified material yielded a major band of the same apparent molecular weight. Factor VII remains bound to the column and, therefore, for subsequent use preincubation of tissue factor with factor VII and calcium is not required. Thus, the present purification procedure markedly reduces the amount of factor VII needed as affinity ligand to purify tissue factor apoprotein.     

The phosphorylation of purified phospholamban by cyclic AMP-dependent protein kinase is stimulated by phosphatidylinositol

A pure bovine phospholamban sample was phosphorylated by cyclic AMP-dependent protein kinase maximally to about 1 mol of phosphate/mol of protein (Mr 25,000), whereas phospholamban purified from bovine cardiac SR (sarcoplasmic reticulum) vesicle prephosphorylated by the protein kinase was found to contain 4.6 mol of phosphate/mol of phospholamban. The decrease in phospholamban phosphorylation occurred during the protein purification at the immunoaffinity chromatography step. The protein phosphorylation could be restored by the addition of the affinity column flow-through fraction to the phosphorylation reaction. The phosphorylation-stimulating activity of the flow-through fraction was resistant to boiling and trypsin treatment and extractable by organic solvent, suggesting that the endogenous factor(s) is lipid. Various phospholipids were found capable of stimulating the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase, but only phosphatidylinositol could stimulate the protein phosphorylation to a level achieved by the phosphorylation of SR membrane-bound phospholamban, about 5 mol of phosphate/mol. Phospholamban phosphorylated in the presence of phosphatidylinositol showed similar sites of phosphorylation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shifts as the phospholamban isolated from phosphorylated SR vesicles. Results of the present study suggest that phospholamban in SR is embedded in a phosphatidylinositol-rich microenvironment, and that this specific environment may be important for the regulation of Ca2+ pump by phospholamban.