Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent

We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.     

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.     

The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2

Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Noncooperative vs. cooperative reactivation of D-beta-hydroxybutyrate dehydrogenase: multiple equilibria for lecithin binding are determined by the physical state (soluble vs. bilayer) and composition of the phospholipids

D-beta-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme that catalyzes the interconversion of beta-hydroxybutyrate and acetoacetate. The purified soluble enzyme devoid of lipid (i.e., the apodehydrogenase) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Lipid activation curves have a sigmoidal shape, and two models have been proposed to explain them. We have previously reported that the kinetics of reactivation with short-chain lecithins in the soluble state is consistent with a model in which the enzyme enzyme contains two identical, noninteracting lecithin binding sites, both of which must be occupied to activate the enzyme [noncooperative mechanism; Cortese, J.D., Vidal, J.C., Churchill, P., McIntyre, J.O., & Fleischer, S. (1982) Biochemistry 21, 3899-3908]. More recently a kinetic model involving cooperative interactions between lecithin binding sites was proposed for the reactivation of the membrane-bound enzyme [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208]. This study reinvestigates the basis for the different conclusions in these two studies. The previous study with soluble lecithins was limited to about 34% of maximal activation compared with mitochondrial phospholipid, due to inactivation of the enzyme at the critical micellar concentration. We could now extend this study to 91% activation by increasing the ethanol concentration. This experimental evidence confirms that the soluble system follows a noncooperative equation. We provide a new kinetic approach to test the cooperative model. A velocity equation is derived for a Hill-type cooperative ligand binding system interacting with a mixture of ligands. This equation predicts a proportionality between an overall weighted cooperative dissociation constant [Kcoop(w)] and a dissociation constant for a single lecithin (PC) species from interacting sites (KPC), regulated by the PC molar fraction (XPC): 1/Kcoop(w) = XPC/KPC. The equation was applied to the data of Sandermann et al. [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208] as well as to newly obtained data. The results obtained over a wide range of PC molar fractions and different mixtures of bilayer phospholipids fit this equation, confirming the cooperative behavior. We conclude that BDH has a different mode of reactivation depending on the nature of the lipid environment. With soluble lecithin, the activation is noncooperative, whereas in the bilayer, mixtures of phospholipids give cooperative behavior that fits a Hill equation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.     

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.     

Spin label study of local anesthetic-lipid membrane interactions. Phase separation of the uncharged form and bilayer micellization by the charged form of tetracaine

The interaction between tetracaine and egg phosphatidylcholine (egg PC) multibilayers was examined. ESR spectra of an ester spin label indicate that at low uncharged anesthetic: lipid ratios, membrane organization decreases. At higher ratios, saturation and phase separation occur, as suggested by a second spectral component which appears when the water solubility of tetracaine is reached. However, experiments with the drug in the absence and in the presence of membranes, making use of a phospholipid spin label, suggest that the new phase does not consist of solid tetracaine alone. Location of the new phase in the membrane would require a change in partition coefficient, while its location outside would imply a mechanism whereby the anesthetic would come off the membrane as an aggregate containing spin probe and phospholipid. Charged tetracaine forms micelles which disrupt-unilamellar egg PC vesicles (Fernandez, M.S. (1981) Biochim. Biophys. Acta 646, 27-30). Micellar tetracaine added to bilayers containing a PC spin probe changes the spectrum from one typical of a bilayer into one typical of micelles, indicating the formation of a tetracaine-egg PC mixed micelle. The effect is reversible upon dilution to concentrations below the critical micelle concentration of tetracaine. When membranes are prepared in the presence of a water-soluble spin label, TEMPOcholine, ascorbate destroys the signal of untrapped label; when mixed phospholipid-tetracaine are formed by addition of micellar tetracaine, this leads to a complete loss of the ESR signal. High drug concentrations are often used for anesthesia and could be related to morphological nerve damage caused by large doses of anesthetics.     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. II. Interaction with phospholipid vesicles

Phosphorylation of endogenous and artificial protein substrates by protein kinase P is stimulated by phosphatidylinositol or phosphatidylglycerol (D. J. Klemm, and L. Elias (1987) J. Biol. Chem. 262, 7580-7585; L. Elias and A. Davis (1985) J. Biol. Chem. 260, 7023-7028). Stimulation of protein kinase P activity required phospholipid vesicles rather than free phospholipid molecules. Protein kinase P activity increased as the phosphatidylinositol content of the vesicles was raised from 20 to 100%; no stimulation was detected below 20% phosphatidylinositol. This suggests that a vesicle surface rich in phosphatidylinositol is required for enzyme activation. Maximum activation of protein kinase P activity showed an optimum value with respect to phospholipid concentration, with both endogenous and artificial protein substrates. The phospholipid concentration at which optimal enzyme activity occurred shifted in response to the concentration of protein substrate, but not enzyme concentration. Therefore, the density of substrate molecules on the surface of phospholipid vesicles is a critical feature of protein kinase P stimulation. Binding of protein kinase P to vesicles was independent of micelle composition, but the binding of the artificial substrate, histone H2B, was specific for vesicles containing phosphatidylinositol or phosphatidylglycerol, and increased as the content of phosphatidylinositol was increased. Thus, an important feature of protein kinase P activation appeared to be the specific binding of protein substrate to phospholipid vesicles.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents

Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.     

Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation.

The activity of phosphocholine cytidylyltransferase (CT), the regulatory enzyme in phosphatidylcholine synthesis, is dependent on lipids. The enzyme, obtained from rat liver cytosol, was purified in the presence of Triton X-100 [Weinhold et al. (1986) J. Biol. Chem. 261, 5104]. The ability of lipids to activate CT when added as Triton mixed micelles was limited to anionic lipids. The relative effectiveness of the lipids tested suggested a dependence on the negative surface charge density of the micelles. The mole percent lipid in the Triton mixed micelle required for activation decreased as the net charge of the lipid varied from 0 to -2. Evidence for the physical association of CT with micelles and vesicles containing phosphatidylglycerol was obtained by gel filtration. The activation by micelles containing PG was influenced by the ionic strength of the medium, with a higher surface charge density required for activation at higher ionic strength. The micelle surface potential required for full activation of CT was calculated to be -43 mV. A specificity toward the structure of the polar group of the acidic lipids was not apparent. CT was activated by neutral lipids such as diacylglycerol or oleyl alcohol when included in an egg PC membrane, but the activities were reduced by dilution with as little as 10 mol % Triton. Thus Triton mixed micelles are not suitable for studying the activation of CT by these neutral lipid activators. We conclude that one way that lipid composition can control CT-membrane binding and activity is by changing the surface potential of the membrane. Other distinct mechanisms involved in the activation by neutral lipids are discussed.     

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.     

The enzyme rhodanese can be reactivated after denaturation in guanidinium chloride

For the first time, the enzyme rhodanese has been refolded after denaturation in guanidinium chloride (GdmHCl). Renaturation was by either (a) direct dilution into the assay, (b) intermediate dilution into buffer, or (c) dialysis followed by concentration and centrifugation. Method (c) preferentially retained active enzyme whose specific activity was 1140 IU/mg, which fell to 898 IU/mg after 6 days. The specific activity of native enzyme is 710 IU/mg. Progress curves were linear for the dialyzed enzyme, and kinetic analysis showed it had the same Km for thiosulfate as the native enzyme, but apparently displayed a higher turnover number. Progress curves for denatured enzyme directly diluted into assay mix showed as many as three phases: a lag during which no product formed; a first order reactivation; and an apparently linear steady state. An induction period was determined by extrapolating the steady-state line to the time axis. The percent reactivation fell to 7% (t1/2 = 10 min) as the time increased between GdmHCl dilution and the start of the assay, independent of the presence of thiosulfate. The induction period, which decreased to zero as the incubation time increased, was retained in the presence of thiosulfate. There were no observable differences between native and renatured protein by electrophoresis or fluorescence spectroscopy. Previous reports of some refolding of urea-denatured rhodanese (Stellwagen, E. (1979) J. Mol. Biol. 135, 217-229) were confirmed, extended, and compared with results using GdmHCl. A working hypothesis is that rhodanese refolding involves intermediates that partition into active and inactive products. These intermediates may result from nucleation of the two rhodanese domains, which exposes hydrophobic surfaces that become the interdomain interface in the correctly folded protein.     

High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction

The number of phosphatidylserine molecules involved in activating protein kinase C was determined in a mixed micelle system where one monomer of protein kinase C binds to one detergent:lipid micelle of fixed composition. Unusually high cooperativity, specificity, and multiplicity in the protein kinase C-phospholipid interaction are demonstrated by examining the lipid dependence of enzymatic activity. The rates of autophosphorylation and substrate (histone) phosphorylation are specifically regulated by the phosphatidylserine content of the micelles. Hill coefficients of 8-11 were calculated for phosphatidylserine-dependent stimulation of enzyme activity, with a maximum occurring in micelles containing greater than or equal to 12 phosphatidylserine molecules. The high specificity that exists is illustrated by the fact that phosphatidylethanolamine and phosphatidylglycerol, but not phosphatidylcholine or phosphatidic acid, can replace only some of the phosphatidylserine molecules. We propose that Ca2+ and acidic phospholipids cause the protein to undergo a conformation change revealing multiple phosphatidylserine binding sites and resulting in the highly cooperative and specific interaction of protein kinase C with phosphatidylserine. Consistent with this, the proteolytic sensitivity of protein kinase C increases approximately 10-fold in the presence of phosphatidylserine and Ca2+ compared to Ca2+ alone. The high degree of cooperativity and specificity may provide a sensitive method for the physiological regulation of protein kinase C by phospholipid.     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.