A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Inhibition of human salivary alpha-amylase by glucopyranosylidene-spiro-thiohydantoin

This study is the first report on the effectiveness and specificity of glucopyranosylidene-spiro-thiohydantoin (G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosyl-maltoside (GalG(2)CNP) hydrolysis catalysed by human salivary alpha-amylase (HSA). The inhibition of hydrolysis is a mixed-noncompetitive type. In any case, only one molecule of inhibitor binds to HSA. Since our substrate and inhibitor are small molecules the long enough active site facilitates accommodating both of them simultaneously. However, the product formation can be excluded from enzyme-substrate-inhibitor complex (ESI) since Dixon plots are linear. Kinetic constants calculated from secondary plots and nonlinear regression are almost entirely equal, confirming the fidelity of the suggested model. Kinetic constants (K(1i)=7.3mM, L(1i)=2.84 mM) show that G-TH is not such a potent inhibitor of HSA as acarbose and indicate higher stability for ESI than for enzyme-inhibitor complex.     

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.     

Contribution of cytosolic cysteine residues to the gating properties of the Kir2.1 inward rectifier

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals (T(c) >or= 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP(2), with C311R and C311S being more potent at modulating the Kir2.1-PIP(2) interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP(2).     

The effect of fructan on membrane lipid organization and dynamics in the dry state

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by (2)H, (31)P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70 degrees C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.     

Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus

Transient electrical currents generated by the Na(+)-transporting F(o)F(1)-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the F(o)F(1)-ATPase that is related to the translocation of the cation through the membrane bound F(o) subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15-30 s(-1) if H(+) is transported and 30-60 s(-1) if Na(+) is transported. For ATP synthesis the rate constant is 50-70 s(-1).     

Role of water-soluble polysaccharides in bacterial cellulose production

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC) and a water-soluble polysaccharide called acetan in corn steep liquor-fructose medium. Acetobacter xylinum EP1, which is incapable of acetan production was derived by disrupting the aceA gene of BPR2001. The BC production by EP1 (2.88 g/L) was lower than that by BPR2001 (4.6 g/L) in baffled-flask culture. When purified acetan or agar was added to the medium from the start of cultivation, the BC production by EP1 was enhanced and the final BC yield of EP1 was almost the same as that of BPR2001. A similar improvement of BC production by EP1 by the addition of agar was also confirmed by cultivation in a 50-L airlift reactor. From these results, the role of acetan in BC production is associated with the increase in the viscosity of the culture medium which may hinder coagulation of BC and cells in the culture, thereby accelerating the growth of BPR2001 and BC production by BPR2001.