Partial characterization and inactivation of membrane-bound phosphofructokinase from Tetrahymena pyriformis

In Tetrahymena pyriformis, 6-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is membrane-bound. Enzyme activity is solubilized by treatment of membranes with Triton X-100 or by high ionic strength in the presence of a chelator. The solubilized enzyme has an approximate molecular weight of 300 000. Both the membrane-bound enzyme and the solubilized enzyme exhibit maximum activity over a wide pH range. At low pH, the membrane-bound form of the enzyme is irreversibly inactivated, whereas the solubilized enzyme is not. The membrane-bound enzyme is inactivated by incubation with Mg2+, ATP, fluoride and a soluble factor that is heat labile, nondialysis, (NH4)2SO4 precipitable and sensitive to trypsin. The solubilized enzyme is not inactivated under similar conditions.     

Properties of digitonin-solubilized calmodulin-dependent guanylate cyclase from the plasma membranes of Tetrahymena pyriformis NT-1 cells

Calmodulin-dependent guanylate cyclase from Tetrahymena plasma membranes was solubilized in about a 22% yield by using digitonin in the presence of 0.2 mM CaCl2 and 20% glycerol. The detergent, when present in the assay at concentrations above 0.05%, diminished the basal and calmodulin-stimulated activity of the enzyme. Guanylate cyclase solubilized with digitonin was eluted from DEAE-cellulose with 200 mM KCl in a yield of 50%. Properties of the solubilized enzyme were similar to those of the native membrane-bound enzyme. The Kms for Mg-GTP and Mn-GTP were 140 and 30 microM, respectively. The enzyme required Mn2+ for maximum activity, the relative activity in the presence of Mg2+ being 30% of the activity with Mn2+. The solubilized enzyme retained the ability to be activated by calmodulin, with its extent being reduced as compared to the membrane-bound enzyme. The presence of a Ca2+-dependent calmodulin-binding site on the solubilized enzyme was shown by the Ca2+-dependent retention of the enzyme on a calmodulin-Sepharose-4B column.     

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.     

Catalytic properties of Candida antarctica lipase B clusters solubilized in hexane

The objective of this work was to investigate the particle size and determine the catalytic competency of a solubilized lipase in hexane. Purified Candida antarctica lipase B (CALB) was solubilized in hexane using the non-ionic surfactant Span 60. The amount of surfactant was chosen so that complete coverage of the individual enzyme molecules with surfactant was not possible. Dynamic Light Scattering (DLS) was used to directly investigate the particle size of the solubilized entities. The enzyme was found to be solubilized in the form of clusters of lipase molecules with a radius of 37±5 nm at 42°C, which we estimate to correspond to about 1200 CALB molecules. The solubilized enzyme clusters showed lower catalytic activity in a model esterification reaction in hexane compared with a commercial immobilizate of the same enzyme (Novozym 435). Further gains in catalytic activity may be possible by striving for true molecular-level dispersion of the enzyme in hexane.     

Solubilization and characterization of particulate form of DNA polymerase from adult rat liver nuclei

DNA polymerase was solubilized from adult liver chromatin-membrane complex. The activity of this solubilized enzyme was 20–30 times higher than that of the partially purified cytoplasmic DNA polymerase. The solubilized nuclear particulate enzyme differed from the cytoplasmic enzyme in properties such as template preference, salt effect and pH optimum. ATP stimulated only the cytoplasmic enzyme, but EDTA and spermidine, stimulated the solubilized nuclear particulate enzyme but not the cytoplasmic enzyme. On sucrose density gradient centrifugation the cytoplasmic DNA polymerase sedimented around 9 S and the solubilized nuclear enzyme sedimented around 3–4 S.     

Activation of solubilized liver membrane adenylate cyclase by nucleotides

Liver plasma membranes isolated from hypophysectomized rats were treated with 0.1 M Lubrol-PX, a nonionic detergent, and centrifuged at 165,000 × g for 1 hour. Adenylate cyclase activity remaining in the supernate had a specific activity that was at least equal to that of the particulate enzyme. The activity of the solubilized, non-sedimentable adenylate cyclase, as well as the membrane bound enzyme, was increased by GTP, ITP, and GMP-PCP at 10^?4 M. The activity of the solubilized, non-sedimentable enzyme increased linearly with GTP from 10^?6 to 10^?4 M but there was no further increase in the activity of the solubilized enzyme with 10^?3 M GTP. In contrast, the particulate liver membrane enzyme activity increased exponentially with GTP from 10^?6 to 10^?4 M and was further increased by 10^?3 M GTP. These data indicate that GTP, ITP or GMP-PCP have direct effects on solubilized adenylate cyclase. This effect is in addition to a role of nucleotides in modifying membrane structure (16).     

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes.

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.     

The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.     

The nitric oxide reductase of Paracoccus denitrificans.

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.     

Identification and solubilization of a cAMP-independent protein kinase from mouse L-cell smooth membranes

1. Smooth membranes have been prepared from mouse L-cells and found to contain an endogenous protein kinase activity. 2. The enzyme(s) responsible for this activity use ATP, but no other nucleoside triphosphates, to phosphorylate endogenous membrane proteins as well as exogenously-added protein substrates such as phosvitin and casein. 3. Mg2+ is required for enzyme activity, maximal activity is observed at pH 7.5-8.0 and the kinase is not dependent on, or stimulated by, cyclic 3'-5' AMP. 4. The kinase activity is not decreased by the Walsh heat-stable inhibitor of cyclic 3'-5' AMP-dependent protein kinases. 5. Fifty percent or more of the membrane-associated kinase activity can be solubilized by extracting membranes with buffer containing 0.6 M NaCl. 6. The solubilized enzyme resembles the membrane-associated activity in its Mg2+ requirement, pH optimum and independence of cyclic 3'-5' AMP. 7. Phosvitin and casein are better exogenous substrates than histones or protamine for phosphorylation by the enzyme in either the membrane-associated or solubilized state.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M (r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always <2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.     

Solubilization of the starch-granule-bound starch synthase of normal maize kernels

The starch-granule-bound starch synthase from Zea mays has been solubilized with a recovery of between 50 and 84%. Chromatography of the solubilized enzyme on DEAE-Sepharose resolves two fractions of activity which may be distinguished by their response to citrate. Neither solubilized isoenzyme displays any significant activity with UDPglucose.     

Regulation of catalytic properties of enzymes in "inverse micelles"]

A triple system (inverse micellae) that simulates the membrane environment of the enzyme was studied. Inverse micellae were obtained using anionic (aerosol OT), synthetic (Brij 56), and natural (lecithin) surfactants. It was found that upon inclusion of an enzyme into inverse micellae, its activity can be regulated by changing the structure and nature of the surfactant matrix. It was shown that enzyme activity in micellar environment is much higher than in water solution. Moreover, the enzyme solubilized in inverse micellae (acid phosphatase) shows a superactivity. It was found that surfactants specifically interact with solubilized enzyme, and the activity of the enzyme is inversely proportional to surfactant concentration. The mechanisms of viscotropic regulation of enzyme activity are discussed.     

The NADPH oxidase of guinea pig polymorphonuclear leucocytes

Summary NADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The K_m value was 15.8 µM for NADPH and 434 µM for NADH indicating that NADPH is the preferential substrate.     

Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells.

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

The effect of bile acids on "solubilized" cholesterol 7 alpha-hydroxylase activity in swine.

Optimal assay conditions for cholesterol 7 alpha-hydroxylase activity in swine liver microsomes were determined. The enzyme activity is induced three-fold by feeding cholestyramine to the swine. This suggests that cholesterol 7 alpha-hydroxylase is likely to be the rate-limiting enzyme for biosynthesis of bile acids in swine. The effects of various bile acids on cholesterol 7 alpha-hydroxylase in swine microsome and "solubilized" cholesterol 7 alpha-hydroxylase activity have been studied. There is no significant reduction of native microsomal enzyme activity. However, except for chenodeoxycholic acid, most of the bile acids tested exerted significant inhibition on "solubilized" cholesterol 7 alpha-hydroxylase. This finding suggests that bile acids could interact with and regulate the rate-limiting enzyme for bile acid formation in swine.     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.     

Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate

In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detergent activation and solubilization of 2'':3''-cyclic nucleotide 3''-phosphodiesterase from isolated myelin and c6 cells.

Several detergents were investigated for their ability to increase activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. The ability of Triton X-100 and Sulfobetaine DLH to solubilize the enzyme was also examined. Solubilization with Triton X-100 was only effective in the presence of salt, for example with NaCl 51% of the activity was solubilized. A single extraction with Sulfobetaine DLH yielded slightly more solubilized enzyme and did not require added salt. Both activation and solubilization of 2':3'-cyclic nucleotide 3'-phosphodiesterase appeared to be similarly dependent on detergent concentration, suggesting a common action of the detergent in the two processes. Myelin basic protein was solubilized more readily than the enzyme. In contrast with the enzyme in myelin, 2':3'-cyclic nucleotide 3'-phosphodiesterase activity in C6 cells was not increased in the presence of Triton X-100, and was partially solubilized by either Triton X-100 or NaCl alone. No myelin basic protein could be detected in C6 cells by radioimmunoassay.