Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.     

Isolation and characterization of fourteen ribosomal proteins from small subunits of yeast.

A method for preparation of a large amount of ribosomal subunits from Saccharomyces cerevisiae by a Ti-15 zonal rotor is described. The proteins of the small subunits (ca. 50 000 A260 units) were separated into 22 fractions by chromatography on carboxymethylcellulose columns. Fourteen proteins were then purified from the ten chromatographic fractions by filtration through Sephadex G-100 or Sephacryl S--200. The isolated proteins are YP 6, YP 7, YP 9, YP 12, YP 14', YP 14', YP 28, YP 38, YP 45, YP 50, YP 52, YP 58, YP 63, and YP 70. The molecular weight and amino acid compositions of these proteins are presented.     

Separation and partial characterization of isolectins with different subunit compositions from Datura stramonium seeds

The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical M_r values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid.     

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

Purification of anthrax-toxin components by high-performance anion-exchange, gel-filtration and hydrophobic-interaction chromatography.

A procedure has been developed for purification of the tripartite anthrax-toxin components. This involves sequential high-performance anion-exchange, gel-filtration and hydrophobic-interaction chromatography. From an initial culture volume of 15 litres, typical yields of 8 mg of protective antigen, 13 mg of lethal factor and 7 mg of oedema factor are produced to higher degrees of purity than have previously been achieved by conventional chromatographic techniques.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Association of leucogenenol,a thymothyroid hormone,with carrier proteins in the thymus

Leucogenenol a heterocyclic enolic thymothyroid hormone (MW 383) whose concentration in the serum regulates the rate at which already committed cells of the bone marrow develop into functional cells, was found to be associated in the thymus with a carrier protein. The carrier protein for leucogenenol is not precipitated by heating to 80° but following this treatment leucogenenol is precipitated in association with proteins precipitated by acetone and then by saturated ammonium sulfate. On chromatography on Sephacryl G-200 it was found that leucogenenol was associated with proteins of MW approximately 38,000. Leucogenenol is not eluted from the chromatographic column if it is not associated with its carrier proteins. It is suggested that other hormones such as those associated with the reproductive cycle or compounds that result from tissue damage induce the liberation of leucogenenol from its carrier protein in the thymus to the circulation where it is associated as previously described, with a protein of approximately MW 300,000.     

Butyl-Toyopearl 650 as a new hydrophobic adsorbent for water-soluble enzyme proteins

Butyl-Toyopearl 650, a butyl derivative of Toyopearl HW-65, was synthesized for use in hydrophobic chromatography. Water-soluble enzyme proteins were adsorbed on butyl-Toyopearl 650 in the presence of ammonium sulfate and eluted easily in the absence of the salt. Cytochrome c, myoglobin, and chymotrypsinogen A were successfully separated on a butyl-Toyopearl 650 column in order of their individual hydrophobicity by decreasing the concentration of ammonium sulfate contained in the buffer eluant. Based on these results, the use of butyl-Toyopearl 650 is demonstrated for the hydrophobic separation of water-soluble enzyme proteins.     

Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants.

The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration behavior of EpiC depended on the oxidation state of the enzyme, indicating the formation of an intramolecular disulfide bridge between C-274 and C-321. The cysteine residues in the motifs WC-274YG and C-321HG of EpiC are conserved in all lantibiotic enzymes of the C type (so-called LanC proteins) and in the CylM protein. Mutated epiC genes from S. epidermidis epiC mutants were cloned and expressed in E. coli. Sequence analysis revealed that the mutations occurred in the two motifs -S-X-X-X-G-X-X-G- and -N-X-G-X-A-H-G-X-X-G-, which are conserved in all LanC proteins. For the investigation of EpiC-EpiA interactions, precursor peptide EpiA was coupled to N-hydroxysuccinimide-activated Sepharose High Performance Material (HiTrap). Under reducing conditions, EpiC was retarded on the EpiA-HiTrap column. In the incubation experiments, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides.     

Properties of albumins of milled rice

Extraction studies on IR36 milled rice showed that albumins solubilized by 0.1–0.15 M (NH₄)₂SO₄ consisted of about 20% high(～5%) lysine, fast-migrating proteins on electrophoresis at pH 8.3 and about 80% lower ～2%) lysine proteins of slower mobility. The 2%-lysine albumins were insoluble in 1.8 M (NH₄)₂SO₄ while the higher lysine albumins required 4 M (NH₄)₂SO₄ to precipitate. The 2%-lysine albumins were not fractionated by gel filtration and gave only one major fraction with MW 19 000. SDS-polyacrylamide gel electrophoresis confirmed the major subunit to be of MW 17 000. These albumins were separated by DEAE-Sephacel chromatography at pH 8.5 into three fractions of similar aminograms but differing in analytical get electrophoretic and isoelectric focusing patterns.     

Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.     

Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by high-performance liquid chromatography and thin-layer chromatography

A fast and sensitive method for the determination of putrescine, spermidine, spermine and ammonia by high-performance liquid chromatography (HPLC) with dabsyl chloride is described. These compounds are converted to their chromophoric dabsyl derivatives and are separated by a normal-phase chromatographic column (μPorasil, 10 μm) with 2% acetone in chloroform as isocratic mobile phase. The sensitivity of the method is 20 pmoles. The present method was shown to be a straightforward procedure for estimating polyamines in various rat tissues.The chromophoric derivatives of polyamines are also well separated by thin-layer chromatography (TLC) on silica gel, and the combination of the HPLC and TLC procedures provides a reliable method for qualitative and quantitative analysis of polyamines.     

Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Some molecular properties of plant starch phosphorylases and the levels of activity of the multiple forms

A number of plant tissues have two phosphorylase fractions (I and II) that can be separated by DEAE-cellulose chromatography. When only one is detectable, it corresponds to enzyme II. Peas differed from other legumes in showing an increase in enzyme I during seed germination. Examination of the I type enzymes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis, sector cell ultracentrifugation and gel filtration, indicated that these were dimers composed of similar sub-units of MW near 90 000. When phosphorylase II enzymes were examined, the sub-unit MW was found to be higher, near 110 000 and, whereas ultracentrifugation techniques indicated a dimer of similar sub-units for the native enzyme, gel filtration gave higher MW values. Phosphorylase II from Victory Freezer peas differed from the other samples, in being able to form mixtures of dimer and tetramer.     

Rapid separation of proteins and their higher-molecular fragments by means of Spheron ion-exchanges.

Ion-exchange derivatives are described. of a hydrophilic rigid macroporous glycolmethacrylate gel called Spheron, suitable for rapid high-performance liquid chromatography (HPLC) of proteins and their fragments. Their flow parameters are compared with those of ion exchange derivatives of cellulose and polydextran. The conditions for work with them are described (regeneration, cycling, equilibration, column packing) as well as the construction of a simple apparatus for medium-pressure ion exchange chromatography of proteins. The efficiency of these ion exchangers for the separation of proteins is illustrated with examples of chromatography of an artificial mixture of serum albumin, chymotrypsinogen and lysozyme. Chromatography of cyanogen bromide fragments of serum albumin and the A and B chains of oxidized insulin showed that the method can be applied in chromatography on higher molecular protein fragments. A review of all proteins, including technical enzymes, which have already been chromatographed on Spheron ion exchangers is also given. The prospects of Spheron ion exchangers for HPLC of proteins and their fragments are briefly discussed.     

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).     

Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucroonate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) functionally heterogeneus; 4-nitrophenol and bilirubin are representative subtrates for two separated from of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine(T₃) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitropheelos was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponing UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T₃ on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.     

Characterization and subcellular localization of vicilin and phytohemagglutinin,the two major reserve proteins of Phaseolus vulgaris L.

Extracts of bean (Phaseolus vulgaris L. cv. Greensleeves) cotyledons contained two abundant proteins: vicilin and phytohemagglutinin. Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had 3 non-identical subunits with molecular weights (MW) of 52,000, 49,000 and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had 2 non-identical subunits with MW of 34,000 and 36,000. Phytohemagglutinin could be separated by isoelectrofocusing into a mitogenic and non-erythroagglutinating protein with a single subunit of MW=34,000, and a mitogenic and erythroagglutinating protein fraction which contained both subunits. Vicilin is apparently identical with the so called glycoprotein II (A. Pusztai and W.B. Watt, Biochim. Biophys. Acta 365, 57–71, 1970) and with globulin G1 (R.C. McLeester, T.C. Hall, S.M. Sun, F.A. Bliss, Phytochem. 2, 85; 1973), while phytohemagglutinin is identical with globulin G2 (McLeester et al., 1973). Since vicilin and phytohemagglutinin are internationally used names there is no need to introduce new names to describe P. vulgaris reserve proteins. Both proteins are catabolized in the course of seedling growth and are located in the protein bodies, indicating that they are reserve proteins. Vicilin isolated in its 18.0 S form from the cotyledons of young seedlings contains substantial quantities of smaller polypeptides, in addition the 3 original ones. We suggest that the presence of these small polypeptides represents partial breakdown of the vicilin prior to its complete catabolism.     

Interactions between cadmium,selenium and glutathione peroxidase in rat testis

In rats given a minimal damaging dose of ¹⁰⁹CdCl₂ (0.011 mmole/kg, s.c.), a visible hemorrhagic response was evident after 48 h when testicular Cd uptake exceeded a level of approx. 150 ng/g. Glutathione peroxidase (GSH-Px) activity was elevated in homogenates of these damaged testes. In rats whose testes were not damaged, the Cd levels were below 150 ng/g and the GSH-Px activity was similar to that of control animals injected with sodium acetate. Rat testis cytosol was found to contain two different GSH-Px activities when assayed with cumene hydroperoxide. These could be separated by gel filtration chromatography. The larger species (GSH-Px A) was eluted in the void volume on Sephadex G-150 and incorporated ⁷⁵Se from Na₂⁷⁵SeO₃ given 4 weeks earlier. The smaller species, of approx. 42 000 molecular weight (MW) (GSH-Px B), did not incorporate ⁷⁵Se and could be distinguished from GSH-Px A by its insensitivity to cyanide (10 mM). CdCl₂ (1 mM) did not inhibit GSH-Px activity when added in vitro to GSH-Px A or B from testicular cytosol, or to purified GSH-Px isolated from ovine erythrocytes. When ¹⁰⁹CdCl₂ was given in vivo to rats injected 4 weeks previously with a tracer dose of Na₂⁷⁵SeO₃ or added in vitro to cytosol prepared from similarly labeled rats, Sephadex G-150 chromatography of cytosol showed that most of the ¹⁰⁹Cd was eluted in a major peak of 34 000 MW. Little or no ¹⁰⁹Cd was found in association with ⁷⁵Se (major peak 140 000 MW) or GSH-Px activity. When ¹⁰⁹CdCl₂ was injected into rats given an equimolar dose of Na₂⁷⁵SeO₃ 30 min previously, ¹⁰⁹Cd uptake in cytosol was increased and both ¹⁰⁹Cd and ⁷⁵Se was shifted into a peak of 110 000 MW.The ¹⁰⁹Cd-binding peak of approx. 30 000–34 000 MW was the major Cd-binding fraction in cytosol of 7-week-old rats but was not detectable in 4-week-old rats. Susceptibility of the testes to Cd did not correlate with the presence of this peak, however, since 4-week-old rats were occassionally damaged by CdCl₂.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase