Riboflavin binding protein contains a type II copper binding site

Riboflavin binding protein, purified from egg white, binds copper(II) under dialysis conditions in an approximately 1:1 molar ratio. Results further indicate a small, but not negligible, amount of copper is present in the protein as purified from egg white. Electron paramagnetic resonance indicates a single type II copper site present in the protein. These results suggest the possibility of a previously unknown function of riboflavin binding protein in the storage or transport of copper.     

Actin co-purifies with RNA polymerase II.

RNA polymerase II, [EC2.7.7.6], from the slime mold Physarum polycephalum, purified over 4000-fold can contain a protein with an apparent molecular weight of 46,000. This protein is separated from the putative subunits of RNA polymerase II by polyacrylamide gel electrophoresis under non-denaturing conditions, and by chromatography on phosphocellulose. In this report we identify the protein as actin, and we point out that polypeptides of this apparent molecular weight which have been found associated with RNA polymerase II purified from other sources may also be actin from these organisms.     

Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.     

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells.

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate. Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I, suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid could be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8, and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydrofolate, lower affinity for N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formyltetrahydrofolate or methotrexate.     

Further comparison of calmodulin-dependent protein kinase II from brain and calmodulin-dependent glycogen synthase kinase from skeletal muscle

Calmodulin-dependent protein kinase II was purified from rabbit brain and its properties were compared with those of calmodulin-dependent protein kinase II from rat brain and calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Rabbit brain calmodulin-dependent protein kinase II was clearly distinguished from rabbit skeletal muscle glycogen synthase kinase with respect to size, behavior on autophosphorylation, immunological cross-reactivity and peptide mapping, but was indistinguishable from rat brain calmodulin-dependent protein kinase II in all respects examined. Thus, differences between calmodulin-dependent protein kinase II and glycogen synthase kinase appear not to reflect a species difference but to reflect a tissue difference.     

Purification and properties of a soluble angiotensin II-binding protein from rabbit liver

An angiotensin II-binding activity has been purified almost 3,000-fold to a nearly homogenous state from the 100,000 x g supernatant fraction of rabbit liver. The responsible protein is apparently monomeric since its molecular weight was estimated to be 75,000 in the native state by glycerol gradient centrifugation and in the reduced, denatured state by gel electrophoresis. The Kd and Bmax values of the purified preparation were 7.2 nM and 15.2 nmol of angiotensin II bound per mg of protein, the latter figure agreeing well with the theoretical value of 13.3. Competition experiments with 125I-angiotensin II and unlabeled peptides revealed that the angiotensin antagonist [Sar1,Ala8]angiotensin II (saralasin) and the agonist [des-Asp1]angiotensin II (angiotensin III) were more tightly bound than angiotensin II, whereas angiotensin I and the carboxyl-terminal hexapeptide were less avidly bound. The cardiac peptide, atrial natriuretic factor, also competed for binding to the purified preparation but was about 15-fold less effective than angiotensin II. Although the binding activity was purified in the absence of detergent, a requirement for detergent in the binding reaction emerged during the isolation procedure. Binding by the purified protein exhibited an almost complete dependence upon the presence of detergent, p-chloromercuriphenylsulfonic acid and EDTA.     

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468–7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833–2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of M_r 42000 and 25000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.     

O-acetylserine sulfhydrylase and S-sulfocysteine synthase activities of Chromatium vinosum

Two proteins containing O-acetylserine sulfhydrylase activity were purified from Chromatium vinosum. Their separation was carried out by DE52 or Ecteola cellulose chromatography. While protein I with a molecular weight of 56,000 had only O-acetylserine sulfhydrylase activity, protein II with a molecular weight of 50,000 possessed S-sulfocysteine synthase activity in addition. It was not possible to separate the two activities of protein II by electrophoretic methods. The reaction rate of protein II with sulfide and O-acetylserine was twice as high as that with thiosulfate and O-acetylserine. When extracts of sulfate-grown cells were purified the major O-acetylserine activity was always associated with protein II. Regulatory and kinetic phenomena of the two activities were studied.     

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: Structural characterization and identification as histidine-rich glycoprotein

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.     

Identification of myosin II as a binding protein to the PH domain of protein kinase B

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.     

Methyl acceptors for protein methylase II from human-erythrocyte membrane.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.     

Glycogen synthesis in the obliquely striated muscle of Ascaris suum

A glycogen synthase, designated GS II, which occurs in a protein/carbohydrate complex has been purified from Ascaris suum muscle. The purified GS-II complex which is eluted from concanavalin-A--Sepharose contains proteins with Mr 140,000 and 66,000 and a glycoprotein with a carbohydrate/protein mass ratio of 3:1. GS II activity was totally dependent on glucose 6-phosphate, but exogenous glycogen was not required for polysaccharide synthesis. The GS-II complex was not phosphorylated by cyclic-AMP-dependent protein kinase, and antibodies to the protein and carbohydrate components of GS II did not cross react with the purified cyclic-AMP-regulated glycogen synthase (GS I) from A. suum muscle. Polysaccharide which was synthesized de novo by the complex was added to the large-molecular-mass glycoprotein in GS II. The glycogen-like character of the newly synthesized polysaccharide was confirmed by the observation that glycogen phosphorylase utilized the polymer as substrate in both the synthesis and degradation reactions. A model is discussed in which a core glycoprotein serves as the substrate for a glycogen synthase which is distinctly different from GS I.     

Purification of protease nexin II from human fibroblasts

Normal human fibroblasts secrete a protein named protease nexin II (PN II) which previously was shown to form sodium dodecyl sulfate (SDS)-stable complexes with epidermal growth factor-binding protein (EGF-BP). These complexes then bind to the same cells and are rapidly internalized and degraded (Knauer, D.J., and Cunningham, D.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2310-2314). Here we describe a procedure for purifying PN II to apparent homogeneity from serum-free culture medium conditioned by human fibroblasts. The first step employed dextran sulfate-Sepharose affinity chromatography. Further purification was achieved by ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-400. Sequence analysis of purified PN II identified 33 amino-terminal amino acids; a computer search of several protein sequence data banks failed to reveal homologies with other reported amino acid sequences. Purified PN II had an apparent Mr of 106,000 and an isoelectric point of approximately 7.2. It retained full activity after incubation in the presence of 0.05% SDS or at a pH of 1.5. PN II formed SDS-stable complexes with EGF-BP, the gamma subunit of 7 S nerve growth factor, and trypsin with estimated Mr of 120,000, 120,000, and 110,000, respectively. PN II was metabolically labeled with [35S]methionine and purified; the metabolically labeled protein formed complexes with EGF-BP. Complexes between purified PN II and EGF-BP bound to human fibroblasts. These results show that the purified protein possesses the properties previously attributed to PN II in cell culture medium.     

Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II.

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.