Activation of protein kinase C with cardiolipin-containing liposomes in relation to membrane-protein interaction

Many cytoplasmic proteins, including Ca2+- and phospholipid-dependent protein kinase (protein kinase C) of polymorphonuclear leukocytes (PMNs) associate in Ca2+-dependent manner with phospholipid liposomes containing cardiolipin (CL), as in the case of phosphatidylserine (PS)-containing liposomes. A crude protein kinase C fraction was purified by association of the enzyme with CL-containing liposomes (flotation method). The partially purified protein kinase C from rat brain or guinea pig PMN was activated by the CL-containing liposomes in the presence of dioleoylglycerol (DG) and Ca2+. This activation was analogous to that of PS. The half maximum activity was obtained with 20 microM CL in the presence of 1 microM Ca2+ and 5 microM DG. Many of the cytoplasmic proteins which associate with CL-containing liposomes were preferentially phosphorylated by membrane-associated protein kinase C in the presence of DG and Ca2+. These results suggest that the association of cytoplasmic protein kinase C with the membrane has an important role in regulation of protein kinase C activity in relation to the association of other cytoplasmic proteins to the membrane.     

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.     

Molecular mechanisms for the induction of peroxidase activity of the cytochrome c-cardiolipin complex

Induction of the peroxidase activity of cytochrome c (cyt c) by cardiolipin (CL) and H(2)O(2) in mitochondria is suggested to be a key event in early apoptosis. Although electrostatic interaction between the positively charged cyt c and negatively charged CL is a predominant force behind the formation of a specific cyt c-CL complex and sequential induction of the peroxidase activity, molecular mechanisms of hydrophobic interactions involving the fatty acyl chains of CL remain to be investigated. To elucidate the function of the acyl chains, particularly the role of the double bond, we synthesized a variety of CL analogues and examined their peroxidase inducing activity. Irrespective of the number of double bonds in the acyl chains, the peroxidase activity of cyt c induced by liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and a different CL (9:1 molar ratio) was similar, except for that of 1,1',2,2'-tetrastearoylcardiolipin (TSCL, C18:0)-containing liposomes. The peroxidase inducing activity of TSCL-containing liposomes was 3-4-fold greater than that of other CL-containing liposomes. The peroxidase activity induced by all CL-containing liposomes was much lower at high ionic strengths than that at low ionic strengths because of diminution of the electrostatic interaction. The peroxidase inducing effects of various CL-containing liposomes were related well to their ability to associate with cyt c. Thus, our results revealed that at low CL levels, the saturated acyl chain of CL is favorable for the activation of peroxidase activity of CL-bound cyt c and the proposed critical role of the double bond is not a general feature of the cyt c-CL interaction. The polarity of the membrane surface of TSCL-containing liposomes was slightly, but significantly, lower than that of other CL-containing liposomes, suggesting that the higher activating ability of TSCL-containing liposomes may be due to a reduced level of hydration of the polar head region reflecting tighter packing of the fully saturated acyl chains. Moreover, using CL analogues in which a central glycerol head moiety was modified, we revealed that the natural structure of the head moiety is not critical for the formation of the active cyt c-CL complex. The effects of the CL content of the liposomal membrane on the cyt c-CL interaction are discussed.     

Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.     

Roles of the N domain of the AAA+ Lon protease in substrate recognition,allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell‐division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross‐linking and scanning for mutations that prevent sul20‐peptide binding. These N‐domain mutations limit the rates of proteolysis of model sul20‐tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon‐mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP‐dependent biological activities do not require translocation.     

Cardiolipin and mitochondrial carriers

Members of the mitochondrial carrier family interact with cardiolipin (CL) as evident from a variety of functional and structural effects. CL stabilises carrier proteins on isolation with detergents, with the P_i carrier as the prime example. CL is required for transport in reconstituted vesicles, prime examples are the P_i- and ADP/ATP carrier (AAC). CL binds to the AAC in a graded manner; 6 CL/AAC dimer bind tightly as measured on the ³¹P NMR time scale. 2 additional CL/dimer bind reversibly and a fast exchanging envelope of phospholipids includes CL as measured on the ESR time scale. In the crystal structure of the CAT-AAC complex 3 CL bind to the periphery of the AAC in a three-fold pseudo-symmetry. The binding of CL is implicated to contribute lowering the high transition energy barriers in the AAC. Para-functions of the AAC, as in the mitochondrial pore transition (MPT) and in cell death are linked to the CL binding of the AAC. Ca⁺⁺ or oxidants can sequester or destroy AAC bound CL, rendering AAC labile, allowing pore formation and degradation. Thus AAC, by being vital for energy transfer, constitutes an Achilles heel in the eukaryotic cell. AAC together with CL is also engaged in respiratory supercomplexes. Different from AAC the similarly structured uncoupling protein (UCP1) has no tightly bound CL, but CL addition lowers affinity of the inhibitory nucleotide binding that may contribute to the physiological regulation of the uncoupling activity by ATP.     

Ca2+-induced increased lipid packing and domain formation in submitochondrial particles. A possible early step in the mechanism of Ca2+-stimulated generation of reactive oxygen species by the respiratory chain.

Ca2+ and P(i) accumulation by mitochondria triggers a number of alterations leading to nonspecific increase in inner membrane permeability [Kowaltowski, A. J., et al. (1996) J. Biol. Chem. 271, 2929-2934]. The molecular nature of the membrane perturbation that precedes oxidative damage is still unknown. EPR spectra of spin probes incorporated in submitochondrial particles (SMP) and in model membranes suggest that Ca(2+)-cardiolipin (CL) complexation plays an important role. Ca(2+)-induced lipid domain formation was detected in SMP but not in mitoplasts, in SMP extracted lipids, or in CL-containing liposomes. The results were interpreted in terms of Ca2+ sequestration of CL tightly bound to membrane proteins, in particular the ADP-ATP carrier, and formation of CL-enriched strongly immobilized clusters in lipid shells next to boundary lipid. The in-plane lipid and protein rearrangement is suggested to cause increased reactive oxygen species production in succinate-supplemented, antimycin A-poisoned SMP, favoring the formation of carbon-centered radicals, detected by EPR spin trapping. Removal of tightly bound CL is also proposed to cause protein aggregation, facilitating intermolecular thiol oxidation. Lipid peroxidation was also monitored by the disappearance of the nitroxide EPR spectrum. The decay was faster for nitroxides in a more hydrophobic environment, and was inhibited by butylated hydroxytoluene, by EGTA, or by substituting Mg2+ for Ca2+. In addition, Ca2+ caused an increase in permeability, evidenced by the release of carboxyfluorescein from respiring SMP. The results strongly support Ca2+ binding to CL as one of the early steps in the molecular mechanism of Ca(2+)-induced nonspecific inner mitochondrial membrane permeabilization.     

Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 μM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK₂ decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

The effects of ATP and sodium chloride on the cytochrome c-cardiolipin interaction: the contrasting behavior of the horse heart and yeast proteins

In cells a portion of cytochrome c (cyt c) (15–20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c–CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)–CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 microM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK2 decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

The different roles of metal ions and water molecules in the recognition and catalyzed hydrolysis of ATP by phenanthroline-containing polyamines

The phenanthroline bridging polyaza ligands L1, L2 and L3 can selectively and strongly bind nucleotides at physiological pH, and hence accelerate the hydrolysis rate of the bound ATP. It is interesting that a phosphoramidate intermediate at 2.88 ppm (should be added 5.63 ppm when compared with other models) was found in the hydrolysis process of L/ATP. By introduction of metal ions (critical Zn(2+) or hard Mg(2+), Ca(2+)) to the L/ATP system, recognition of the anionic substrates by the protonated ligands was greatly promoted. However, due to the different affinities of metal ions to the receptor and the substrate, ATP hydrolysis in Zn(2+)/L/ATP system and Mg(2+)(Ca(2+))/L/ATP system occurs through different mechanisms. By comparison with the M/ATP (M=Zn(2+), Mg(2+), Ca(2+)) system, the rates of ATP-hydrolysis in the Mg(2+)Ca(2+)/L/ATP system and the Zn(2+)/L/ATP system were enhanced and retarded, respectively. Moreover, the reasons contributing to large rate range of the L/ATP systems and M(2+)/L/ATP systems were given. The results show that metal ions vertically regulate the recognition and hydrolysis of ATP. On the other hand, water molecule participates in the hydrolysis reactions at different steps with different functions in the L/ATP systems and M(Zn(2+), Mg(2+), Ca(2+))L/ATP systems.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Interaction of secretory protein precursors with phospholipids in liposomes

The precursors of secretory proteins were synthesized in a reticulocyte lysate system programmed with rat serum albumin or human placental lactogen mRNA and their interaction with phospholipids in liposomes was studied. The precursor proteins could bind to acidic phospholipids that have an exposed phosphate such as dicetyl phosphate and phosphatidic acid or a phosphate that is covered by a small moiety such as phosphatidylglycerol. The binding of precursor proteins was dependent on the mol% of acidic phospholipids in lecithin-liposomes, increased with elevation of temperature in the range of 0 to 45 degrees C, and was not inhibited by the addition of a large excess of mature proteins. Mature proteins or proalbumin showed no significant binding to the liposomes containing acidic phospholipids. About 15% of the acid-precipitable radioactivity bound to the liposomes was resistant to protease digestion. This radioactivity was shown to correspond to methionine-containing peptides with molecular weights of 2,500 to 3,500. These results indicate that the post-translational insertion of a small part of the precursor proteins into the membrane did occur with the present model system, but the post-translational transfer of precursor proteins across the membrane did not.     

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease

Lon is an oligomeric serine protease whose proteolytic activity is mediated by ATP hydrolysis. Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites that hydrolyze ATP at drastically different rates. The catalytic low-affinity sites display pre-steady-state burst kinetics and hydrolyze ATP prior to peptide cleavage. The high-affinity sites are able to hydrolyze ATP at a very slow rate. By utilizing the differing Kd's, the high-affinity site can be blocked with unlabeled nucleotide while the activity at the low-affinity site is monitored. Little kinetic data are available that describe microscopic events along the reaction pathway of Lon. In this study we utilize MANT-ATP, a fluorescent analogue of ATP, to monitor the rate constants for binding of ATP as well as the release of ADP from Escherichia coli Lon protease. All of the adenine nucleotides tested bound to Lon on the order of 10(5) M(-1) s(-1), and the previously proposed conformational change associated with nucleotide binding was also detected. On the basis of the data obtained in this study we propose that the rate of ADP release is slightly different for the two ATPase sites. As the model peptide substrate [S2; YRGITCSGRQK(Bz)] [Thomas-Wohlever, J., and Lee, I. (2002) Biochemistry 41, 9418-9425] or the protein substrate casein affects only the steady-state ATPase activity of the low-affinity sites, we propose that Lon adopts a different form after its first turnover as an ATP-dependent protease. Based on the obtained rate constants, a revised kinetic model is presented for ATPase activity in Lon protease in both the absence and presence of the model peptide substrate (S2).     

Characterization of a glucosyltransferase activity in liver plasma membrane: modulation by cations and lipidic effectors

1. Glucosyltransferase activity incorporating [14C]glucose from UDP-[14C]glucose onto endogenous lipidic acceptors was localized primarily in the plasma membrane of liver. 2. Incubation of plasma membrane by phosphatidyl-choline liposomes loaded with dolichyl-phosphate stimulated the enzymatic activity. 3. This enzyme required Mg2+ for maximal catalitic activity. Ca2+ could substitute Mg2+. 4. Mn2+ acted as a partial non-competitive inhibitor of the Mg2+-activated glucosyltransferase. 5. This enzyme can be modulated by neutral and acidic phospholipids; the most efficient were phosphatidyl-serine and phosphatidyl-inositol. 6. The enzymatic activity was not significantly changed by cholesterol alone but it is greatly enhanced by liposomes loaded with dolichyl-phosphate and cholesterol.     

Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid.

A group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), are the major proteins found in bovine seminal fluid. These proteins are secretory products of seminal vesicles, and they bind to spermatozoa upon ejaculation, suggesting that there are binding sites for these proteins on the spermatozoa. It was of interest to characterize these binding sites on spermatozoa which may help in the elucidation of the biological function of BSP proteins. The binding sites on spermatozoa are resistant to protease or acid treatment and are heat-stable but extractable with organic solvents. The solvent-extractable material, when coated on plastic microtitration wells, binds radiolabeled BSP proteins thus indicating the lipid nature of the BSP binding sites on spermatozoa. We investigated the specificity of interaction of BSP proteins with lipids using liposomes of phospholipids, solid-phase, and thin-layer chromatography-overlay techniques. Results showed that BSP-A1, -A2, and -A3 proteins bound specifically to those phospholipids which contain the phosphorylcholine group. In contrast, BSP-30-kDa protein preferentially bound to phospholipids containing the phosphorylcholine moiety but also interacted with phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Furthermore, of those lipids that were extracted from spermatozoa, only phospholipids which contain the phosphorylcholine moiety bound radiolabeled BSP proteins. These data suggest that the BSP protein binding sites on spermatozoa are phospholipids. We propose that this specific interaction plays an important role in the membrane modification of spermatozoa that occurs during capacitation and/or acrosome reaction.