Abnormal neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia

Neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia was isolated under conditions designed to minimize proteolysis. Those methods yielded an α₂β₂ form of myeloperoxidase from normal individuals. Purified enzyme from the patient had electronic absorbances ($\text{A}{}_{430}\text{A}{}_{280}\text{= 0.85}$), enzymatic activity, and electrophoretic and Chromatographic behavior indistinguishable from that of normal myeloperoxidase. Edman degradation and physical studies after reduction and denaturation, however, showed that as compared to normal enzyme, one 55,000-dalton α subunit of the patient's myeloperoxidase was replaced by a 39,000-dalton peptide with a different amino-terminal sequence, a mixture of smaller peptides, and an heme derivative. Myeloperoxidase from the leukemic neutrophils appeared to have been partially degraded in vivo by lysosomal proteases.     

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-M_r protein band as the monomer of the purified enzyme.     

Molecular weights of glycollate oxidase from C3 and C4 plants determined during early stages of purification

The M_rs of glycollate oxidase (EC 1.1.3.1) (GAO) determined soon after extraction from the leaves of several C₃ and C₄ plants are reported. The enzyme isolated from the C₃ plants wheat, barley, spinach, pea and tobacco has M_r in the range 160–180 000 and is probably a homotetramer. GAO purified from pea was previously reported as a dimer and as an octamer from spinach leaves. Therefore the quaternary structure of these GAOs soon after extraction differs from that of the purified proteins. The enzymes from the C₄ plants maize and sugar cane have M_rs ca twice this value in the range 290–310 000, whilst that of the C₄ grass Panicum maximum has an M_r of 162 000. An improved spectrophotometric assay for GAO, using a non-carcinogenic dye, is described.     

Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph,Nitrobacter agilis ATCC 14123

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M_r) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M_r values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.     

The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high $\text{pH}\text{6.8}\text{pH}\text{8.2}$ activity ratio (0.4 – 0.7) and a high level of Ca²⁺ independent activity $\text{(EGTA}\text{Ca}{}^{\mathrm{2+}}\text{= 0.3?0.5)}$. The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a M_r 128,000 polypeptide in the gradients. This polypeptide and a M_r 143,000 polypeptide were labeled with ³²P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a M_r 102,000 membrane polypeptide which followed the distribution of Ca²⁺-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A M_r 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca²⁺/calmodulin-stimulated manner, including a M_r ca. 72,000 polypeptide found only in the transverse tubule.     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate

A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The M_r of the purified enzyme is 95,000 with two nonidentical subunits of M_r 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of M_r 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis.     

Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a M_r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M_r of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and K_a values of 67 nM and 1.1 μM for calmodulin and Ca²⁺, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca²⁺, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.     

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP⁺ 2-oxidoreductase (decar☐ylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of M_r 70 000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2′-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.     

A comparison of the membrane-bound and extracellular cyclic AMP phosphodiesterases of Dictyostelium discoideum

The Dictyostelium discoideum membrane-bound and extracellular cyclic nucleotide phosphodiesterases (EC 3.1.4.17) shear several properties including the ability to react with a specific glycoprotein inhibitor and small inhibitory molecules. We have partialy purified the membrane-bound enzyme and compared its properties to those of the extracellular form. The kinetic properties of the two forms were similar except that, while associated with membrane particles, the membrane-bound form exhibited non-linear kinetics when assayed ove a broad substrate range. The isoelectric point of the membrane-bound phosphodiesterase was identical to that of the extracellular enzyme when isoelectrofocusing was done in the presence of 6 M urea. The molecular weights of membrane-bound and extracellular enzyme, determined by gel filtration, were the same following isoelectrofocusing in the presence of 6 M urea. When precipitated with an antiserum prepared against purified extracellular phosphodiesterase, the partially purified membrane-bound enzyme preparation was shown to contain a M_r 50 000 polypeptide comigrating with the extracellular enzyme during SDS polyacrylamide gel electrophoresis. When the iodinated extracellular enzyme and the iodinated M_r 50 000 polypeptide from membrane-bound enzyme were subjected to partial proteolytic digestion, similar profiles were obtained indicating extensive regions of homology.     

Purification and properties of a transketolase responsible for formaldehyde fixation in a methanol-utilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201

Dihydroxyacetone synthase, present in methanol-grown Candida boidinii (Kloeckera sp.) No. 2201, catalyzes the transfer of the glycolaldehyde group from xylulose 5-phosphate to formaldehyde to form glyceraldehyde 3-phosphate and dihydroxyacetone. This enzyme was purified to electrophoretic homogeneity and found to be a new type of transketolase. The molecular weight of the enzyme was estimated to be 190 000 by gel filtration. The enzyme appeared to be composed of four identical subunits (M_r, 55 000). Thiamin pyrophosphate and Mg²⁺ were required for the activity. The optimum pH was found to be 7.0. With xylulose 5-phosphate as the ketol-donor, aliphatic aldehydes (C₁?C₇), glycolaldehyde and glyceraldehyde were better acceptors than ribose 5-phosphate. The kinetic data were consistent with a ping-pong bi-bi mechanism. The K_m values obtained were as follows: xylulose 5-phosphate, 1.0 nM; formaldehyde, 0.43 mM; glyceraldehyde 3-phosphate, 0.42 mM; and dihydroxyacetone, 0.52 mM.     

Purification of the activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense

The activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense has been purified to near homogeneity. The procedure includes ion-exchange chromatography, chromatofocusing and gel filtration. The M_r of the purified enzyme was determined to be 33 500 on SDS-polyacrylamide gel electrophoresis. The purified enzyme was compared with the acticating enzyme from Rhodospirillum rubrum.     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

An 88 amino acid long C-terminal sequence of human lactotransferrin

The terminal oxidoreductase of nitrous oxide respiration in the marine, denitrifying bacterium, Pseudomonas perfectomarinus, was identified as multi-copper protein and purified to electrophoretic homogeneity. The enzyme reduced N₂O to N₂ with hydrogen, clostridial hydrogenase, and methyl viologen as electron-donating system. The copper content of the reductase corresponded to ～ 8 copper atoms/120 000 M_r. The subunit structure was dimeric with two peptides of equal size. Manganese, iron and zinc were absent, or were not found in stoichiometric amounts. The oxidized chromophore had absorption maxima at 350, 480, 530, 620 and 780 nm; addition of dithionite produced a blue protein form with maxima at 470, 635 and 740 nm. Both forms of the enzyme were paramagnetic. The same copper protein was also isolated from Pseudomonas stutzeri.     

Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

A hysteretic invertase from Equisetum giganteum L

The invertase from Equisetum giganteum L., a lower vascular sporophytic plant, was purified to chromatographic and electrophoretic homogeneity. The enzyme appears to be a pentamer, M_r 91,000, formed by identical subunits (M_r 18,000). An isoelectric point of 4.5 was found for the protein. The optimum pH was about 4.5 and the preferred substrate is sucrose, K_m=10.4 mM. Glucose and fructose are classical non-competitive (K_i=120 mM) and competitive (K_i=96 mM) inhibitors, respectively. Proteins which behave as activators of the enzyme suppress the inhibitory action of the reaction products. The activation energy of the hydrolytic reaction is 18,000 cal/mol. The outstanding property of the invertase is a hysteretic behavior when the pH changes from 3.05 to 4.5. The lag time is independent of the enzyme concentration suggesting that slow conformational changes are induced by pH variation and not by different polymerization states.     

Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of UL15-Encoded Proteins with B Capsids Requires at Least the UL6, UL17, and UL28 Genes

The U_L15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U_L15-encoded protein recognized three proteins with apparent M_rs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-M_r protein was detected in type C capsids and comigrated with the product of a U_L15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-M_r proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔU_L15), a virus lacking an intact U_L15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of U_L15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent M_rs of 83,000, 80,000, and 79,000 are products of U_L15 with identical C termini. The 79,000-, 80,000-, and 83,000-M_r proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U_L15-encoded proteins are integral components of B capsids. Only the 83,000-M_r protein was detected in B capsids purified from cells infected with viruses lacking the U_L6, U_L17, or U_L28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-M_r proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-M_r U_L15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.     

Diamine oxidase ofLathyrus sativus seedlings. Purification and properties

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings ofLathyrus sativus by MnCl₂ treatment, (NH₄)₂SO₄ and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had anM _r of 148,000 (6.46S) and was a dimer with similar sub-units (M _r 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the K_m for benzyl amine without any change in the V_max Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to theL. sativus enzyme.