The effects of phospholipids on the properties of hepatic 5'-nucleotidase

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.     

Physiological role and membrane lipid modulation of the membrane-bound (Mg2+, na+)-adenosine triphosphatase activity in Acholeplasma laidlawii.

The membrane-bound adenosine triphosphatase (ATPase) activity of Acholeplasma laidlawii B differs in many respects from the common (Mg2+, Ca2+)-ATPase activity of higher bacteria, most notably in that it is specifically activated by Mg2+ and strongly and specifically stimulated by Na+ (or Li+). Various inhibitors diminish the ATPase activity with a concentration dependence which suggests that a single enzyme species is responsible for all of the observed ATP hydrolytic activity (both basal and Na+ stimulated). The Km for ATP is influenced by temperature but not by membrane lipid fatty acid composition. Vmax is influenced by both of these factors, showing a break in Arrhenius plots which falls below the lipid phase transition midpoint but well above the lower boundary when a phase transition occurs within the temperature range studied. The apparent energy of activation for Vmax is strongly influenced by lipid fatty acid composition both above and below the break. When whole cells of A. laidlawii B are incubated in KCl or NaCl buffers, they rapidly swell and lyse if deprived of an energy source or treated with ATPase inhibitors at concentrations which significantly inhibit enzyme activity in isolated membranes, whereas in sucrose or MgSO4 buffers of equal osmolarity, the cells are stable under these conditions. These results suggest that the membrane ATPase of A. laidlawii B is intimately associated with the membrane lipids and that it functions as a monovalent cation pump which regulates intracellular osmolarity as the (Na+, K+)-ATPase does in eucaryotes.     

Wheat mitochondria: oxidative activity and membrane lipid structure as a function of temperature

Mitochondrial oxidative activity and membrane lipid structure of two wheat (Triticum aestivum L.) cultivars were measured as a function of temperature. The Arrhenius activation energy for the oxidation of both succinate and α-ketoglutarate was constant over the temperature range of 3 to 27 C. The activation energy for succinate-cytochrome c oxidoreductase activity was also constant over the same temperature range. The concentration of mitochondria in the reaction, the degree of initial inhibition of state 3 respiration, and the time after isolation of mitochondria were each shown to be capable of causing a disproportionate decrease in the rate of oxidation at low temperatures which resulted in an apparent increase in the activation energy of oxidative activity. Using three spin-labeling techniques, wheat membrane lipids were shown to undergo phase changes at about 0 C and 30 C. It is concluded that the membrane lipids of wheat, a chillingresistant plant, undergo a phase transition similar to the transition observed in the membrane lipids of chilling-sensitive plants. For wheat, however, the transition is initiated at a lower temperature and extends over a wider temperature range.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

Dechlorination of pentachlorophenol by membrane bound enzymes of Rhodococcus chlorophenolicus PCP-I

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P₄₅₀. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Response of Tonoplast and Plasma Membrane ATPases in Chilling-Sensitive and -Insensitive Rice (Oryza sativa L.) Culture Cells to Low Temperature

Tonoplast and plasma membrane vesicles were prepared from chilling-sensitive(CS) and chilling-insensitive (CI) cultured cells of rice (Oryzasativa L.) to examine how they would respond to low temperature.With CS cells, the specific activity of ATPase in tonoplastvesicles was relatively higher than that of plasma membraneATPase. Tonoplast ATPase activity was decreased by low temperaturetreatment, and a slight decrease in plasma membrane ATPase activitywas also observed. The decrease in the specific activity ofthe tonoplast ATPase by low temperature may reflect a decreasein V_max. However, no change was noted in K_m. The break pointof the Arrhenius plots of the tonoplast ATPase was ca. 32?C,this value being ca. 9?C higher than that of the plasma membraneATPase. With CI cells, the specific activity of tonoplast ATPasewas somewhat less than that of the plasma membrane ATPase. TonoplastATPase activity was decreased by low temperature at 5?C, whereasan increase in plasma membrane ATPase activity was observed.The break point of the tonoplast ATPase activity was ca. 22?C,which was 3?C higher than that of the plasma membrane ATPase.Using ATPase solubilized from the plasma membrane or tonoplast,the Arrhenius plots of log ATPase activity against the reciprocalof absolute temperature gave a straight line fit from 5?C to45?C with no obvious break point. The break point appeared onadding a phospholipid mixture (asolectin) to a reaction mixturecontaining solubilized enzyme. The slope of the curve of theArrhenius plot was very different between the CS and CI cells.The plasma membrane and tonoplast ATPases from the CS cellshad a higher E_a above 20?C, whereas that from the CI cells hada lower one. These findings indicate that the tonoplast ATPase in a riceplant is more sensitive to low temperature than the plasma membraneATPase, with this response possibly being due to interactionsbetween the proteins and phospholipids. (Received January 6, 1988; Accepted July 5, 1988)     

Modification of transmembrane electron transport activity in plasma membranes of simian virus 40 transformed pineal cells

Changes have been found in the plasma membrane enzyme system which carries out transmembrane electron transport and associated proton transport in Simian virus 40 (SV40) temperature-sensitive A (tsA) mutant-transformed rat pineal cell line, RPN209-1. This cell line was temperature-sensitive for the maintenance of transformation. RPN209-1 cells expressed the transformed phenotype (rapid growth, high cell density, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, and lower cloning efficiency in soft agar) at the nonpermissive temperature (40 degrees C). The reduction of external ferricyanide, hexaammine ruthenium and diferric transferrin was used to measure the transmembrane redox activity. The transformed RPN209-1 cells expressed a lower transmembrane redox activity, which is more sensitive to the antitumor drug adriamycin, when compared to the cells with a nontransformed phenotype. The lower transmembrane redox activity is associated with a decrease in the affinity for ferricyanide and a change in Vmax of the enzyme. Since the transformed cells have 25% lower concentration of NADH, the decrease in Vmax may be partly based on substrate limitation. Ionic strength variation in the assay media shows that the change in activity with transformation is not based on change in cell-surface change. Treatment with neuraminidase, however, indicates that sialic acid is important for enzyme activity, consistent with previous proposals that the transmembrane enzyme is a glycoprotein. The proton extrusion associated with transplasma membrane electron transport is increased in transformed cells relative to the rate of ferricyanide reduction. A relation between proton pumping transplasma membrane electron transport and growth stimulation by external oxidants is discussed.     

PROPERTIES OF MEMBRANE-BOUND DOPAMINES-HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA

Abstract— Properties of membrane-bound and soluble dopamine-β-hydroxylase were studied. Both enzyme forms have identical affinities for tyramine as the substrate. Arrhenius plots of the membrane-bound activity displayed a discontinuity at 29°C, the activation energy changing from 20,500 cal/mol below 29°C to 9500 cal/mol above 29°C. The soluble enzyme, like the purified enzyme, did not show discontinuities in Arrhenius plots, the activation energies being 18,500 cal/mol and 16,500 cal/mol respectively. The membrane-bound enzyme showed a discontinuity in the p K^m for tyramine versus reciprocal temperature plot, with a transition at 29°C, whereas the soluble enzyme failed to show such transition.
The membrane-bound dopamine-β-hydroxylase is solubilized by Triton X-100 as well as by lysolecithin. Of lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce solubilization of membrane-bound dopamines β-hydroxylase. The 29°C-transition was not removed by treatment either with lysolecithin or with Triton X-100 used at concentration of up to 2%. This could indicate a solubilization of dopamine-β-hydroxylase with an associated lipid moiety which cannot be dispersed from the enzyme molecule and which still affects the activation energy and Michaelis constant for tyramine.
Results are discussed in terms of the relationship between lipid organization and enzyme activities; the significance of the solubilization by lysolecithin during exocytosis is considered in terms of the exocytosis process and fate of the chromaffin granule.     

Pentachlorophenol-induced change of zeta-potential and gel-to-fluid transition temperature in model lecithin membranes

We have determined zeta-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The zeta-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in zeta-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pretransition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pretransition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in delta Tt is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the delta Tt from photometric studies with the density of adsorbed PCP derived from zeta-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.     

Energy-dependent activation and magnesium--dependent inactivation of hepatocyte hormone-sensitive phosphodiesterase

Incubation of solubilized hormone-activated phosphodiesterase from isolated hepatocytes, under conditions likely to favour a dephosphorylation reaction, did not cause a loss of the hormone activation. If, however, the enzyme was incubated with Mg2+ (10 mM) while still associated with its membrane, and subsequently solubilized, the activity of the hormone-stimulated enzyme declined to the level seen in control cells. Diminution of hepatocyte ATP levels to about 20% of control values, by incubation with fructose, blunted the effect of glucagon and abolished the effect of insulin on phosphodiesterase. More severe ATP depletion caused by dinitrophenol abolished the stimulation of the enzyme by both hormones. These effects were not considered likely to be due to altered hormone-binding and are consistent with the involvement of an energy-dependent step in the hormonal activation of phosphodiesterase.     

Conformational properties of membrane-bound fumarate reductase of Escherichia coli

Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 over-produce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B. D. Lemire, J. J. Robinson, and J. H. Weiner (1982) J. Bacteriol. 152, 1126-1131). Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements. Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in alpha helix and that above pH 11 the structure abruptly changes to a beta-like conformation. The same phenomenon is observed in samples solubilized in the nonionic detergent C12E9. Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane. Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C12E9 produced a decrease in ellipticity and in enzymatic activity.     

Energy-dependent activation and magnesium-dependent in activation of hepatocyte hormone-sensitive phosphodiesterase

Incubation of solubilized hormone-activated phosphodiesterase from isolated hepatocytes, under conditions likely to favour a dephosphorylation reaction, did not cause a loss of the hormone activation. If, however, the enzyme was incubated with Mg²⁺ (10 mM) while still associated with its membrane, and subsequently solubilized, the activity of the hormone-stimulated enzyme declined to the level seen in control cells.Diminution of hepatocyte ATP levels to about 20% of contol values, by incubation with fructose, blunted the effect of glucagon and abolished the effect of insulin on phosphodiesterase. More severe ATP depletion caused by dinitrophenol abolished the stimulation of the enzyme by both hormones. These effects were not considered likely to be due to altered hormone-binding and are consistent with the involvement of an energy-dependent step in the hormonal activation of phosphodiesterase.     

Changes in thermodependence of the tyrosine transaminating activity in mitochondria of Drosophila melanogaster larvae due to environmental stress

1. The form of Arrhenius plots of enzyme in mitochondria isolated from Drosophila melanogaster larvae exposed to heat shock, ethanol, or ethanol and heat shock, solubilized with charged detergents was analysed. 2. Heat shock and ethanol caused different changes in membrane microenvironment of the tyrosine transaminating activity, which found expression in different forms of Arrhenius plots, and different values of activation energy of enzyme. 3. The Arrhenius plots of the enzyme from mitochondria of larvae exposed both to ethanol and heat shock, solubilized with charged detergents, were similar to those observed for mitochondria from organisms exposed only to ethanol.     

Concanavalin A induced activity change in yeast PM-bound NADH-HCF(III) oxidoreductase

The activity of plasma membrane bound redox enzyme, NADH-HCF(III) oxidoreductase, in wild and mutant strains of the yeast Saccharomyces cerevisiae is modulated by Con A in a dose-dependent manner. The solubilized activity is enhanced at lower concentration and inhibited at higher concentration of Con A. The enzyme in mutant strain is more sensitive to inhibition. The activation of enzyme by Con A is suppressed in the presence of either alpha-methyl-D-mannoside or 2-deoxy-D-glucose, indicating the glycoproteic nature of enzyme as well as the resulting conformational change due to interaction with Con A as the factor for modulated activities. This was supported by recording the decrease in K(m) value of enzyme with respect to substrate NADH in the presence of lower concentration of Con A. The purified enzyme was more sensitive to lectin stimulation and, on the basis of comparative stimulatory effects of Con A and PSA on activity, it is likely that mannosyl moiety in enzyme is involved in binding the lectins to cause enzymic activation.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

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.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Adsorption capacity as a key parameter for enzyme induction and pentachlorophenol degradation in Mycobacterium chlorophenolicum PCP-1.

Adsorption of pentachlorophenol (PCP) on induced cells of Mycobacterium chlorophenolicum PCP-1 and its influence on enzyme induction and PCP degradation of this strain were studied. Compared to non-induced cells, induced degrading cells had a lower adsorption capacity (q(ads)), particularly at prolonged induction and low PCP concentration. Unlike the effects of pH and biomass concentration previously reported for non-induced cells, the variation of q(ads) of induced cells was associated with changes of both the capacity and intensity constants of the Freundlich equation which was used to describe PCP adsorption on M. chlorophenolicum PCP-1. This indicated changes of cell surface properties during enzyme induction and PCP degradation. The latter was shown in turn to be affected by several parameters such as PCP concentration, pH value and induction time. Interestingly, irrespective of the pH and PCP concentration, the specific PCP degradation rate (q(t)(PCP)) at a given induction time was found to be solely a function of q(ads), revealing that adsorption capacity is an inherent key parameter for enzyme induction and PCP degradation. Based on this knowledge, a kinetic model was developed for q(t)(PCP) which used only q(ads) and induction time as variables. The model considered inhibition of PCP on both enzyme induction and enzyme activity and described the experimental data at different PCP concentrations and pH values well. q(ads) also turned out to be a useful criterion for choosing optimum induction concentration of PCP. Irrespective of pH and biomass concentration, an initial adsorption capacity of 2-3 micromol PCP/g cells was found to be optimum for enzyme induction in M. chlorophenolicum PCP-1.     

The function and activity of certain membrane enzymes when localized on – and off – the membrane

A group of enzymes known to be involved in group translocation-type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt-Ozer and Stadtman, 1971 a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P-ribose-PP as an intermediate; the enzyme in aqueous solution requires P-ribose-PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypo-xanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, 1976) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reflect the in situ capacities of the enzyme and the gain of guanine specificity was determined in a guanine PRT deletion strain (Jackman and Hochstadt, 1976). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30-fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which “appears” largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because the majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.