Organization of microsomal UDP-glucuronosyltransferase. Activation by treatment at high pressure

Treatment of microsomes at pressures as high as 2.25 kbar led to an apparent irreversible activation of UDP-glucuronylsyltransferase when pressure was released. The response of the enzyme to pressure, as reflected by activity measured after release of pressure, appeared to be discontinuous in that no activation was seen for any preparation at pressures less than 1.2 kbar. In addition, activation was temperature dependent. Maximum activation at 2.25 kbar occurred at about 12 degrees C; the extent of activation in 10 min was less for either higher or lower temperatures. Activation was also time dependent. Maximum activation at 2.25 kbar and 9 degrees C required 90 min of pressure treatment. Activation appeared to occur more slowly at lower pressure. Pressure-induced activation was associated with a loss of sensitivity of the enzyme to allosteric activation by UDP-N-Ac-Glc and a conversion of the kinetic pattern from non-Michaelis-Menten to Michaelis-Menten. Pressure did not activate enzyme that had previously been activated maximally by adding detergent to microsomes. Pressure also did not activate pure UDP-glucuronosyltransferase reconstituted into unilamellar vesicles of dioleoylphosphatidylcholine. Pressure treatment did not release UDP-glucuronosyltransferase from microsomes into water. Pressure had a continuous effect on the polarization and excimer/monomer formation of fluorescent probes incorporated into microsomes, and the properties returned essentially to their values at 1 atm when pressure was released. Measurements of activity at 2.2 kbar showed that pressure-induced activation of UDP-glucuronosyltransferase in microsomes occurred via two intermediates that were inactive and that the activated state of the enzyme was generated during/after release of pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH

Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6-P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-6-P was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, e.g. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo.     

Effects of high pressure on the catalytic and regulatory properties of UDP-glucuronosyltransferase in intact microsomes.

The effects of high pressure on the kinetic properties of microsomal UDP-glucuronosyltransferase (assayed with 1-naphthol as aglycon) were studied in the range of 0.001-2.2 kbar to clarify further the basis for regulating this enzyme in untreated microsomes. Activity changed in a discontinuous manner as a function of pressure. Activation occurred at pressure as low as 0.1 kbar, reaching one of two maxima at 0.2 kbar. As pressure was increased above 0.2 kbar, activity decreased, reaching a minimum at about 1.4 kbar followed by a second activation. The pathway for activation at pressure greater than 1.4 kbar was complex. The immediate effect of 2.2 kbar was nearly complete inhibition of activity. The inhibited state relaxed, however, over about 10 min (at 10 degrees C), to a state that was activated as compared with enzyme at 0.001 kbar or enzyme at pressures between 1.4 and 2.2 kbar, which was the highest pressure we could test. Examination of the detailed kinetic properties of UDP-glucuronosyltransferase indicated that the effects of pressure were due to selective stabilization of unique functional states of the enzyme at 0.2 and 2.2 kbar. Activation at 0.2 kbar was reversible when pressure was released. This was true as well as for activation at pressure greater than 1.4 kbar, but after prolonged treatment at 2.2 kbar, UDP-glucuronosyltransferase became activated irreversibly on release of pressure. The process by which prolonged treatment at 2.2 kbar led to permanent activation of UDP-glucuronosyltransferase after release of pressure was not reflected, however, by time-dependent changes in the functional state of UDP-glucuronosyltransferase at this pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholesterol on the function and thermotropic properties of pure UDP-glucuronosyltransferase

The effects of cholesterol on the activity and thermal properties of a pure, delipidated isoform of UDP-glucuronosyltransferase were examined after incorporation of enzyme into unilamellar bilayers of distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC). Cholesterol, in bilayers of DSPC, decreased enzyme activity and lowered the temperature (from 37 to 30 degrees C) for a reversible transition from the active form of the enzyme to a less active form. These effects could be separated from each other in that the effect on reversible inactivation of the enzyme occurred at lower concentrations of cholesterol than the effect on activity of the active form of the enzyme. In addition, cholesterol in bilayers of DSPC stabilized UDP-glucuronosyltransferase against irreversible thermal inactivation. The extent of stabilization increased with increasing concentration of cholesterol in the bilayers. The effects of cholesterol on UDP-glucuronosyltransferase depended, however, on the nature of the bilayer containing cholesterol. Cholesterol had small effects, if any, on the properties of UDP-glucuronosyltransferase in bilayers of DOPC.     

Absence of hepatic p-nitrophenol UDP-glucuronosyltransferase induction by spironolactone in male rats: possible involvement of testosterone.

This study was performed to determine whether the lack of spironolactone induction of hepatic p-nitrophenol UDP-glucuronosyltransferase in male rats could be attributed to a presumed interaction between spironolactone and testosterone. The effect of spironolactone was evaluated in four experimental groups: normal females, normal males, castrated males, and castrated males that received testosterone. Enzyme activity was measured in native microsomes and in microsomes activated with UDP-N-acetylglucosamine or Triton X-100. When the nucleotide was included in the incubations, it was observed that enzyme activity in castrated male rats decreased to values approaching those obtained in normal females. Treatment of castrated animals with testosterone enhanced enzyme activity so that no significant difference existed between this group and normal males. This suggests that testosterone may act as an endogenous inducer of hepatic p-nitrophenol glucuronidation. It was also found that only females and castrated males showed an increase in enzyme activity in response to spironolactone treatment. Thus, the absence of an additive effect of endogenous or exogenous testosterone and spironolactone on UDP-glucuronosyltransferase activity suggests that these compounds could share a common induction mechanism, which appears to reach its maximal capacity in male rats. Possible explanations of this observation are discussed. From the analysis of enzyme activity in native and Triton X-100 activated microsomes, it can be postulated that spironolactone enzyme induction in female and castrated male rats could be attributed to an enhancement in the transferase synthesis rather than to an alteration of the membrane environment.     

Factors affecting the activity of the lactate dehydrognease of Streptococcus cremoris

Studies with partially purified extracts of the nicotinamide adenine dinucleotide-linked l(+)-lactate dehydrogenase of Streptococcus cremoris US3 showed that fructose-1,6-diphosphate (FDP) was essential for the catalytic reduction of pyruvate in the pH range 5.0 to 7.0, outside of which the organism does not grow. In the absence of FDP, enzyme activity was observed only in the region of pH 8.0. The optimal pH for the oxidation of lactate was approximately 8.0 in the presence and absence of FDP. The FDP-activated enzyme was markedly inhibited by inorganic phosphate. The enzyme lost activity on standing at 5 C in alkaline triethanolamine, was quite stable at pH 6.0 to 6.5, and underwent irreversible denaturation below pH 5.0. Inorganic phosphate or FDP increased the stability of the enzyme in alkaline buffers. Some distinguishing properties of individual lactate dehydrogenases, activated by FDP, are discussed.     

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.     

Kinetic and allosteric properties of highly-purified, biosynthetic L-threonine dehydrogenase from brewer's yeast Saccharomyces carlsbergensis

Kinetic and allosteric propeties of highly purified "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlbergensis were studied at three pH values, using L-threonine and L-serine as substrates. It was shown that the plot of the initial reaction rate (v) versus initial substrate concentrations ([S]0 pH 6.5 is hyperbolic (Km=5.0.10-2M), while these at pH 7.8 and 9.5 have a faintly pronounced sigmoidal shape with fast occurring saturation plateaus ([S]0.5= 1.0.10-2 and 0.9.10-2M, respectively). the ratios between L-threonine and L-serine dehydratation rates depend on pH. The kinetic properties and the dependence of substrate specificity on pH suggest that the enzyme molecule undergoes pH-induced (at pH 7.0) conformational changes. The determination of pK values of the enzyme functional groups involved in L-threonine binding demonstrated that these groups have pK is approximately equal to 7.5 and 9.5. The latter group was hypothetically identified as a epsilon-NH2-group of the lysine residue. High concentrations of the allosteric inhibitor (L-isoleucine) decrease the rates of L-threonine and L-serine dehydratation and induce the appearance (at pH 6.5) or increase (at pH 7.9 and 9.5) of homotropic cooperative interactions between the active sites in the course of L-threonine dehydratation. The enzyme inhibition by L-isoleucine increases with a decrease of L-threonine concentrations. Low L-isoleucine concentrations, as well as the enzyme activator (L-valine) stimulate the enzyme at non-saturating substrate concentrations (when L-threonine or L-serine are used as substrates) without normalization of (v) versus [S]0 plots. The maximal activation of the enzyme is observed at pHG 8.5--9.0. It is assumed that the molecule of "biosynthetic" L-threonine dehydratase from brewer's yeast contains two types of sites responsible for the effector binding, i.e., "activatory" and "inhibitory" ones.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.     

Purification and characterization of the heat-labile alpha-amylase secreted by the psychrophilic bacterium TAC 240B.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment.     

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.     

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.     

Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate

Polidocanol-solubilized alkaline phosphatase was purified to homogeneity with a specific activity of 822.3 U/mg. In the absence of Mg2+ and Ca2+ ions and at pH 9.4, the enzyme hydrolyzed ATP in a manner that could be represented by biphasic curves with V = 94.3 U/mg, K0.5 = 17.2 microM, and n = 1.8 and V = 430.3 U/mg, K0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity sites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the specific activity increased to as much as 1,000 U/mg, whereas the K0.5 and n values remained almost unchanged. In the presence of nonsaturating concentrations of metal ions, the hydrolysis of ATP was similar to that observed in the absence of these ions, but with a marked decrease in K0.5 values. At pH 7.5, the enzyme also hydrolyzed ATP with K0.5 = 8.1 microM and V = 719.8 U/mg. Apparently, alkaline phosphatase was able to hydrolyze ATP in vivo, either at pH 7.5 or pH 9.4. These data contribute to the knowledge of the biological properties of skeletal alkaline phosphatase and suggest that this enzyme may have a high-affinity binding site for ATP at alkaline pH.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

Partial purification of microsomal signal peptidase from hen oviduct

Signal peptidase has been purified approximately 600-fold from hen oviduct microsomes. Treatment of microsomes with ice-cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P-40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.