Hydroxylapatite Column Research Articles

Partial purification and properties of a glucosyltransferase that synthesizes Glc1Man9(GlcNAc)2-pyrophosphoryldolichol.

The glucosyltransferase that transfers the first glucose residue from dolichyl-P-glucose to Man9-(GlcNAc)2-PP-dolichol has been solubilized from porcine aorta and purified 720-fold. The purification strategy involved ammonium sulfate precipitation followed by ion-exchange, gel filtration, and hydroxylapatite column chromatographies. Analysis of the products produced by enzyme fractions at different stages of purification indicate that three different glucosyltransferases are involved in the conversion of Man9(GlcNAc)2-PP-dolichol to Glc3Man9(GlcNAc)2PP-dolichol. the first glucosyltransferase appears to be specific for dolichyl-P-glucose as the donor substrate. Man9(GlcNAc)2-PP-dolichol, Man7(GlcNAc)2-PP-dolichol, and Man5(GlcNAc)2-PP-dolichol (with two different oligosaccharide structures) were tested for their ability to accept glucose from dolichyl-P-glucose. Studies on the comparative rates of transfer of glucose to these different acceptor substrates demonstrated that Man9(GlcNAc)2-PP-dolichol accepts glucose at a higher initial rate and to a greater extent than does Man7(GlcNAc)2-PP-dolichol and the biosynthetic Man5(GlcNAc)2-PP-dolichol. The other Man5(GlcNAc)2-PP-dolichol (i.e. Man alpha 1,6[Man alpha 1,3]-Man alpha 1,6[Man alpha 1,3]Man beta 1,4GlcNAc beta 1, GlcNAc) was not an acceptor, indicating that the Man alpha 1,2-Man alpha 1,2Man alpha 1,3Man arm is necessary. Man9(Glc-NAc)2 and Man9(GlcNAc)2-protein were not acceptors, indicating that both the lipid and the oligosaccharide portion of Man9(GlcNAc)2-PP-dolichol are required for enzyme activity. The partially purified enzyme has a pH optimum of 6.5 and exhibits a requirement for divalent metal ions.

Purification and characterization of two isoforms of serine proteinase from the microsomal membranes of rat liver.

An extensive purification of microsomal serine proteinase was achieved from rat liver microsome membranes solubilized with 1% sodium cholate by a series of column chromatographic steps on hydroxylapatite, DEAE-cellulose, and benzamidine-Sepharose 6B, and polyacrylamide gel electrophoresis (PAGE). In the final step of PAGE, the enzyme was separated into two fractions with slightly different mobilities, designated microsomal serine proteinases 1 (MSP1) and 2 (MSP2). The former was purified about 8,000-fold to apparent homogeneity, and the latter was purified about 800-fold, starting from the sample which had been spontaneously activated after elution from the hydroxylapatite column. Both enzyme fractions showed essentially the same properties, including molecular weight, susceptibility to various proteinase inhibitors and metal ions, specificity of action toward protein substrates with special preference for a basic protein histone as well as toward synthetic substrates, and pH dependence of activity toward a synthetic substrate and two neuropeptides. Taken together with the same behavior on a series of chromatographic steps, the two isoforms are thought to be essentially the same enzyme. Further, the detailed kinetic studies of the enzyme activity, especially of MSP1, toward various synthetic and naturally occurring peptide substrates showed that it was highly specific for basic amino acid pairs, strictly hydrolyzing at the COOH side of arginine residue. These results are consistent with those obtained previously with a partially purified enzyme and establish more definitely and in detail the specificity as well as other molecular and enzymatic properties of the enzyme.

Transient activation of a distinct serine protein kinase is responsible for 27-kDa heat shock protein phosphorylation in mitogen-stimulated and heat-shocked cells.

We have investigated the phosphorylation of HSP27, a 27-kDa heat shock protein which is involved in cellular thermoresistance and is also an early target of phosphorylation during heat shock and cell stimulation by a variety of growth and differentiation factors. HSP27 is transiently phosphorylated after shifting Chinese hamster cells from their normal temperature of 37 to 44 degrees C. The phosphorylation correlated in time with the transient activation of specific HSP27 protein kinase activities. HSP27 kinase was also induced to maximal levels within 5-15 min following stimulation of quiescent cells with heat shock, serum, thrombin, or basic fibroblast growth factor. Extracts from quiescent cells stimulated by heat shock or serum were analyzed after sequential chromatography on cation exchange and hydroxylapatite columns. In both cases, a single and identical peak of HSP27 kinase activity was obtained, suggesting that the same protein kinase was induced. The HSP27 kinase efficiently phosphorylated recombinant Chinese hamster HSP27 or the synthetic peptide RALNRQLSSGV containing the major in vivo phosphorylation site of rodent HSP27. The kinase was inactive toward the ribosomal S6 protein, the peptide RALSSLRA from S6 protein, or the mutant HSP27 proteins with in vivo phosphorylation sites altered to glycine. The partially purified HSP27 kinase had no kinase C, kinase A, or S6 kinase activities; conversely, HSP27 was not a good substrate for these kinases. HSP27 kinase was rapidly inactivated in the presence of acid phosphatase, suggesting that its activity was regulated by phosphorylation. It is suggested that this heat shock- and serum-induced HSP27 kinase is a novel serine kinase which is linked to a major signal transduction cascade.

Properties of polyphosphatase of Acinetobacter johnsonii 210A.

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to Pi, was purified 77-fold from Acinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase of Acinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg2+ by pyro- and triphosphate. The apparent Km-value for polyphosphate with an average chain length of 64 residues was 5.9 microM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg2+ and K+.

Purification and Characterization of a Novel Esterase That Hydrolyzes Cannabinoid-11-hydroxyesters from Mouse Hepatic Microsomes

A novel membrane-bound esterase was purified from hepatic microsomes of male ddN mice using ω-aminooctyl Sepharose-4B, DEAE-5PW, CM-Sephadex C-50 and/or hydroxylapatite column chromatographies. The purified enzyme (ES46.5K) showed a single band on a sodium dodecylsulfate-polyacrylamide gel with a mimimum molecular weight of 46.5 Kd. The N-terminal amino acid sequence of the enzyme had no homology to those of known carboxylesterases. ES46.5K exhibited esterase activities toward 11-acetoxy derivatives of tetrahydrocannabinol (THC), whereas the enzyme did not hydrolyze phenolic acetates of cannabinoids (Δ8-THC, Δ9-THC and cannabidiol) except for 1-acetyl-cannabinol. ES 46.5K also possessed esterase activity toward p-nitrophenylacetate, 1-naphtylacetate and 2-acetylaminofluorene. Emulgen 911 and deoxycholate affected differently on the esterase activity of ES46.5K and carboxylesterase purified from porcine liver. Immuno-reactive protein to antibody against ES46.5K was present in hepatic microsomes from mice and rabbits, but not those from rats, guinea pigs and monkeys. The present study demonstrates that mouse hepatic microsomes contain a novel type of esterase having molecular weight of 46.5 Kd and high catalytic activity toward 11-hydroxy-esters of cannabinoids.

Isolation and Characterization of the Tricarboxylate Transporter from Pea Mitochondria

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [(14)C]citrate for internal citrate or malate but not for preloaded d,l-isocitrate. Similarly, although external malate, succinate, and citrate competed with [(14)C]citrate in the exchange reaction, d,l-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less active than the transporter isolated from animal tissues.

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A(615-630) indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 mumol of nitrite formed per min per mg of protein. The cytochrome-containing NR(I) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa alpha-subunit, a 64-kDa beta-subunit, and a 23-kDa gamma-subunit with relative band intensities indicative of a 1:1:1 alpha/beta/gamma subunit ratio and a M(r) of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.

A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.

Immune CD4+ T cells specific for Theileria parva-infected lymphocytes recognize a 24-kilodalton protein.

Theileria parva is a protozoan parasite that infects and transforms bovine lymphocytes. Here we report the partial purification of a T. parva-specific protein from infected lymphocytes that is recognized by CD4+ parasite-specific T-cell clones derived from immune cattle. T. parva-infected lymphocytes were homogenized in Dulbecco's phosphate-buffered saline in the presence of protease inhibitors. The antigen was purified from a postmicrosomal supernatant by using a combination of DEAE-cellulose chromatography and hydroxylapatite column chromatography. After labelling with 125I, the antigen preparation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain 8 to 10 proteins. This preparation was subjected to chromatography in phosphate-buffered saline on HPLC TSK-250/125 columns coupled in tandem. A radiolabelled protein of M(r) 24,000 correlated with antigenic activity.

Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies

We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free α subunit of hCG (hCGα) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits ( E < 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.

Ca 2+-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca 2+-dependent association of the enzyme to the microsomes

Ca 2+-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE—Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca 2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The K m values for ATP and casein were 11 μ m and 300 μg/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48 80 , but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca 2+ and was eluted by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca 2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca 2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca 2+-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.

Biological actions of monoclonal antibodies to bovine lutropin receptor

Monoclonal antibodies (Mabs) were produced against bovine Lutropin receptor (LH-R). Antibodies were detected by an enzyme linked immunosorbant assay (ELISA). Hybridomas were subcloned to achieve monoclonality. Ascites were developed in Balb/c mice. Hybridoma supernatants were purified by ammonium sulfate precipitation and chromatography on hydroxylapatite columns. LH-R antibodies showed upto 50% inhibition of 125I-labeled hCG binding to bovine luteal cell membranes and up to 80% inhibition of testosterone production by hCG stimulated mouse Leydig cells. LH-R antibodies were predominantly IgM isotype. Purified antibodies showed a 78-kDa band, in SDS-PAGE, as the heavy chain of the immunoglobulin. LH-R antibodies were localized specifically in the thecal and luteal cells of the rat ovaries as well as in the Leydig cell of mouse testes. Injections of the LH-R antibody caused a constant estrus in normal rats. One month after the cessation of the injections the animals returned to normal estrus cycle and fertility. Pregnant mice injected with LH-R antibodies produced only 3 viable pregnancies and 10 pups, as compared to 8 pregnancies and 45 pups born to normal controls. LH-R antibodies also caused, approximately, a 50% reduction in testosterone production in normal male rats. These observations indicate a high degree of specificity of the Mab to LH-R and their potential in studies on gonadal function.

Restoration of malonyl-CoA sensitivity of soluble rat liver mitochondria carnitine palmitoyltransferase by reconstitution with a partially purified malonyl-CoA binding protein

Solubilization of rat liver mitochondria in 5% Triton X-100 followed by chromatography on a hydroxylapatite column resulted in the identification of malonyl-CoA binding protein(s) distinct from a major carnitine palmitoyltransferase activity peak. Further purification of the malonyl-CoA binding protein(s) on an acyl-CoA affinity column followed by sodium dodecyl sulfate gel electrophoresis indicated proteins with M r mass of 90 and 45−33 kDa. A purified liver malonyl-CoA binding fraction, which was devoid of carnitine palmitoyltransferase, and a soluble malonyl-CoA-insensitive carnitine palmitoyltransferase were reconstituted by dialysis in a liposome system. The enzyme activity in the reconstituted system was decreased by 50% in the presence of 100 μ m malonyl-CoA. Rat liver mitochondria carnitine palmitoyltransferase may be composed of an easily dissociable catalytic unit and a malonyl-CoA sensitivity conferring regulatory component.

FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.

A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate.

A thiocyanate hydrolase that catalyzes the first step in thiocyanate degradation was purified to homogeneity from Thiobacillus thioparus, an obligate chemolithotrophic eubacterium metabolizing thiocyanate to sulfate as an energy source. The thiocyanate hydrolase was purified 52-fold by steps involving ammonium sulfate precipitation, DEAE-Sephacel column chromatography, and hydroxylapatite column chromatography. The enzyme hydrolyzed 1 mol of thiocyanate to form 1 mol of carbonyl sulfide and 1 mol of ammonia as follows: SCN- + 2H2O----COS + NH3 + OH-. This is the first report describing the hydrolysis of thiocyanate to carbonyl sulfide by an enzyme. The enzyme had a molecular mass of 126 kDa and was composed of three different subunits: alpha (19 kDa), beta (23 kDa), and gamma (32 kDa). The enzyme exhibited optimal activities at pH 7.5-8.0 and at temperatures ranging from 30 to 40 degrees C. The Km value for thiocyanate was approximately 11 mM. Immunoblot analysis with polyclonal antibodies against the purified enzyme suggested that it was induced in T. thioparus cells when the cells were grown with thiocyanate.

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.

Purification and molecular properties of 2-carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver.

1. A rat liver cytosol enzyme, tentatively named CBA reductase, catalyses the conversion of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid in the presence of NADH (or NADPH). CBA reductase is useful for exploring the mechanism of in vitro enzyme induction, as the enzyme can be induced by phenobarbital (PB) both in vivo and in vitro. 2. Possible involvement of glutathione (GSH) in gene expression was suggested by a recent study with cultured rat hepatocytes. 3. CBA reductase was purified about 200-fold by a combination of column chromatography and isoelectric focusing in the presence of mercaptoethanol. 4. The ability to form 2-hydroxymethyl benzoic acid was lost when the enzyme was chromatographed on a hydroxylapatite column in the absence of mercaptoethanol; however, it was restored if sulphydryl compounds or bovine serum albumin was added to the eluate from the column. 5. Gel filtration showed the molecular sizes of CBA reductase from the 105,000g supernatant fraction of rat liver extracts and the purified preparation were 64 kDa and 49 kDa, respectively. 6. The results suggest that sulphydryl substances and some proteins play important roles in preserving the molecular and catalytic properties of CBA reductase.

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.

Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization.

An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.