Resolution and properties of a protein kinase catalyzing the phosphorylation of a wheat germ cytokinin-binding protein

The major cytokinin binding protein of wheat germ (CBP) was extensively purified employing chromatography on Cibacron F3GA-Sepharose CL6B and concanavalin A-agarose as key purification steps. The major polypeptides present in the purified CBP preparations have molecular weights of 60,000 ± 4,000, 42,000 ± 3,000, and 37,000 ± 3,000, respectively. A protein kinase that catalyzes the phosphorylation of CBP (CBP kinase) was extensively purified from wheat germ by affinity chromatography on casein-Sepharose 4B and CBP-Sepharose 4B. The purification procedure resolves CBP kinase from an abundant casein kinase that does not phosphorylate CBP. CBP kinase catalyzes the phosphorylation of casein, phosvitin, CBP, and the wheat germ cyclic AMP-binding protein cABPII. CBP kinase phosphorylates the major 60,000 dalton subunit of CBP as well as 16,000 to 18,000 dalton polypeptides present in CBP preparations. CBP fractions with differing activities as substrates for CBP kinase were partly resolved by gel filtration and by chromatography on DEAE-Sephacel.     

Purification of a Soluble Cytockinin-binding Protein from Etiolated Mung Bean Seedlings

A cytokinin-binding protein (CBP) was purified from a crude extract of etiolated mung bean seedlings by a protocol involving affinity chromatography on benzyladenine-linked Sepharose 4B, ion exchange chromatography on DEAE-Sephadex A50, and gel filtration on Sphacryl S-400. The molecular weight was estimatd to be about 200,000 by gel filtration. CBP appeared as two bands corresponding to molecular weights of about 45,000 and 48,000 on SDS-polyacrylamide gel electrophoresis. The dissociation constant for benzyladenine was 7.5 x 10^-7 M. ¹⁴C-Benzyladenine-binding to CBP was reversible and could be inhibited by the addition of kinetin or trans-zeatin. Adenine, AMP, and ADP had no inhibitory effect on the binding of ¹⁴C-benzyladenine to CBP but the addition of ATP to the assay mixture enhanced the binding.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Partial purification and characterization of the proteins from the 40-S ribosomes of Artemia salina and wheat germ.

The proteins were extracted from purified 40-S ribosomes derived from wheat germ and Artemia salina and separated by carboxymethylcellulose ion-exchange chromatography. Approximately four proteins from Artemia and four proteins from wheat germ were separated in a state of high purity. All proteins were identified by co-electrophoresis using a two-dimensional polyacrylamide gel system. A total of 30 unique proteins were found for Artemia and 32 proteins for wheat. The molecular weights of all proteins were estimated by sodium dodecylsulfate gel electrophoresis. Assuming each protein to be present in one copy per 40-S ribosome, the total protein molecular weight was estimated to be 560,000 associated with Artemia 40-S particles and 550,000 associated with wheat germ 40-S ribosomes.     

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose C1 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca²⁺/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase V1.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Increase in beta-N-Acetylglucosaminidase Activity during Germination of Cotton Seeds

A marked increase in beta-acetylglucosaminidase (2-acetamido-2-deoxy-beta-d-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) activity was observed in the germinating cotyledon of cotton seeds. The enzyme was isolated from cotton seedlings and purified to study its physiological function in the germination of cotton seeds. The purification procedure involves ammonium sulfate fractionation, ion-exchange chromatography, gel filtrations, and concanavalin A-Sepharose 4B chromatography, and the purified beta-N-acetylglucosaminidase was shown to be homogeneous by disc electrophoresis. The molecular weight was estimated to be about 125,000 by gel filtration. The enzyme hydrolyzed both p-nitrophenyl-N-acetyl-beta-d-glucosamine and p-nitrophenyl-N-acetyl-beta-d-galactosamine. When p-nitrophenyl-N-acetyl-beta-d-glucosamine was used as substrate, K(m) and V(max) were 0.625 nanomolar and 228 moles per minute per milligram, respectively, and optimum activity was at pH 5.6. The enzyme liberated beta-linked N-acetyl-glucosamine from chitin, ovalbumin, and pronase-digested wheat germ lectin.     

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.     

Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca²⁺, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K_M of 450 micromolar for ATP and a V_max of 25 picomoles of ³²P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K_i 150 micromolar).     

Purification of the creatine kinase isozymes of the green sunfish (Lepomis cyanellus) with Blue Sepharose CL-6B.

Three homodimeric creatine kinase isozymes (A₂, B₂, and C₂) of the green sunfish (Lepomis cyanellus) were purified by a combination of affinity chromatography, gel filtration, and preparative starch gel electrophoresis. The final preparations were isozymically pure and were used to elicit antibodies in rabbits. The use of the group-specific adsorbant Blue Sepharose CL-6B (Pharmacia) and specific elution conditions for creatine kinase facilitated purification. Fish creatine kinase isozymes are sensitive to denaturation and cannot be readily purified by procedures routinely used for mammalian creatine kinase isozymes.     

Purification of insulin receptor with full binding activity

Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions.     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

Post- -globulin: isolation and physicochemical characterization

Post-γ-globulin has been purified from urine of kidney transplant patients by a procedure which includes ultrafiltration, gel filtration, and ion-exchange chromatography. The purified protein appears to be homogeneous by ultracentrifugal and gel electrophoretic criteria. The yield of the purified protein was 30–35% of the total amount of post-γ-globulin excreted in different urine samples.Electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol revealed a single band and established that the protein consists of one polypeptide unit with a molecular weight of approximately 11,500. Sedimentation velocity experiments indicated a s_{20, w} of 1.55 S. Amino acid analysis of post-γ-globulin revealed the presence of hydroxylysine and hydroxyproline. The protein is devoid of carbohydrate. Post-γ-globulin has been demonstrated in small quantities in normal urine, serum, cerebrospinal fluid, and mixed saliva; preparations derived from these fluids have been shown to be immunochemically identical.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito

The protein demonstrating larvicidal activity to the mosquito Aedes aegypti was purified from the alkali extract of the spore-parasporal inclusion complex of the isolate, 73-E-10-2, belonging to Bacillus thuringiensis serotype 10. By Sepharose CL-4B gel filtration and DEAE-cellulose column chromatography, a toxic protein was obtained, and its homogeneity was confirmed by Sephadex G-150 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the toxic protein was 67,000, when estimated by SDS-PAGE. The LC₅₀ of the toxic protein against 4-day-old larvae of A. aegypti was 16.8 μg/ml. There was no serological relationship between the toxic protein from the isolate 73-E-10-2 and that (M_r 67,000) from the type strain of B. thuringiensis subsp. israelensis.     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Resolution and Properties of Two High Affinity Cyclic Adenosine 3':5'-monophosphate-Binding Proteins from Wheat Germ

A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 × 10⁵ (at low ionic strength) and 2.8 × 10⁵ (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMP-binding protein (cABP I). The K_d values for cAMP are about 10⁻⁶ molar and 10⁻⁷ molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatives having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP.     

Comparison of cytokinin-binding proteins from wheat and oat grains

Cytokinin-binding proteins (CBPs) isolated from mature grains of oat ( Avena sativa L.) and wheat ( Triticum aestivum L.) by acid precipitation, ion-exchange and affinity chromatography had similar characteristics, although they differed somewhat in apparent molecular weight of the native protein as determined by gel filtration (109 and 133 kDa, respectively) and subunit size as estimated by SDS-polyacrylamide gel electrophoresis (47 and 55 kDa, respectively). Highly purified oat CBP showed very weak but distinct immunochemical cross-reactivity with anti-wheat CBP IgG, indicating different immunogenic properties of the two CBPs. Nevertheless, both CBPs exhibited very similar binding of different cytokinins and were characterized by high affinity for N⁶-benzyladenine (BA)-type and by low affinity for zeatin-type cytokinins to both wheat and oat CBPs and by somewhat higher binding activities of oat CBP compared to wheat CBP (K_ds for BA: 4.6 × 10⁻⁷  M and 6.8 × 10⁻⁷  M , respectively). The potential role of CBPs in regulating free BA-type cytokinin levels during cereal grain development and germination is discussed.     

Purification of an antibacterial protein in the coelomic fluid of Nereis diversicolor (annelida,polychaeta). similitude with a cadmium-binding protein

1. Coelomic fluid of Nereis diversicolor shows antibacterial activity 24 hr after inoculation with Escherichia coll, while fluid from untreated control does not.2. This activity is detected by growth inhibition of E. coli or Micrococcus kristinae.3. The thermolabile protein has been purified by two distinct methods using gel filtration and exchange ion chromatography. By SDS-PAGE, the molecular weight of this protein has been estimated at about 10 kDa.4. The protein shows strong similitude with cadmium-binding protein (CBP) which is a dimer constituted by two 10 kDa subunits (Nejmeddine et al., 1988).5. In Western Blot, monoclonal antibody against CBP reacted with the antibacterial protein.6. Moreover, bacteriostatic activity of the coelomic fluid disappeared after incubation with polyclonal and monoclonal antibodies.