The succinic dehydrogenase of seedlings

Crystalline lysozyme has been interacted with an anionic, a cationic, and two nonionic surface-active agents (SAA). Quantitative precipitation of lysozyme by the ionic SAA used was obtained at ratios of the reactants consonant with the formation of stoichiometric complexes dependent upon salt linkages between the SAA and the oppositely charged groups in the enzyme. Neither of the nonionic SAA tested caused precipitation of the enzyme.The inactivation of lysozyme is shown to be constant over a 50-fold range of enzyme concentration when calculated on the basis of the ratio of SAA to enzyme. Inhibition of lysozyme activity as a result of interaction with ionic SAA was obtained only when the ionic SAA were present in substantial excess of the amount required for formation of stoichiometric complexes with oppositely charged groups in the enzyme. Neither of the two nonionic SAA studied altered the enzymatic activity of lysozyme.     

Na(+)-dependent transport of anionic amino acids by preimplantation mouse blastocysts.

Negatively charged amino acids, such as aspartate and glutamate, were selected as substrates by low- and high-Km components of mediated Na(+)-dependent transport in preimplantation mouse blastocysts. These and other relatively small anionic amino acids with two carbon atoms between the negatively charged groups (or up to three carbon atoms when the groups were both carboxyl groups) interacted strongly with the low-Km component of transport, whereas larger anionic amino acids interacted weakly or not at all. The low-Km system was also stereoselective except in the case of aspartate. Moreover, transport was Cl(-)-dependent and slower at pH values outside the range 5.6-7.4. L-Aspartate, D-aspartate and L-glutamate each interacted strongly with the low-Km component of transport with Km values for transport nearly equal to their Ki values for inhibition of transport of one of the other amino acids. By these criteria, the low-Km component of transport of anionic amino acids in blastocysts appears to be the same as the familiar system X-AG that is present in other types of mammalian cells. In contrast, the high-Km component of transport in blastocysts preferred L-aspartate to L-glutamate, whereas the reverse is true for fibroblasts. Therefore, transport of anionic amino acids in blastocysts may occur via at least one process that has not been described in other types of cells. Roughly half of mediated glutamate and aspartate transport in blastocysts may occur via the high-Km component of transport at the concentrations of these amino acids that may be present in uterine secretions.     

Biophysical studies of the interactions between the phage ϕKZ gp144 lytic transglycosylase and model membranes

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage ϕKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.     

Anionic center of porcine pancreatic phospholipase A2

The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions.     

Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting

Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.     

Separation and comparative characterization of the cationic protease and anionic protease from the culture medium of Thermoactinomyces vulgaris

The anionic protease component which frequently contaminates preparations of routinely isolated cationic protease (thermitase) from Thermoactinomyces vulgaris was purified, virtually to homogeneity, by rechromatography on controlled pore glass (CPG-10). Starting materials were column eluates with anionic protease, contaminated with residual thermitase activity. The purified anionic enzyme shares several properties with thermitase, such as size, sensitivity against phenylmethanesulfonyl fluoride and Hg2+, UV-spectral, immunological and pH behavior. On the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable) and enzymatic activity (less active) of anionic protease differ significantly from thermitase. At pH 8 or 6 and 25 degrees or 4 degrees C anionic protease is hydrolysed completely by thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer for thermitase. In contrast to thermitase, the anionic enzyme partially changes spontaneously during long-term storage at 4 degrees C and pH 6 to a cationic protein species endowed with proteolytic activity.     

Characterization of an anionic trypsin from the eel (Anguilla japonica)

An enzyme, isolated from the pancreas of the eel Anguilla japonica and designated as anionic trypsin 1, had a molecular weight of 26,000 and an isoelectric point of 5.5. The amino acid composition of the enzyme was similar to that of bovine cationic trypsin as well as anionic trypsins from other species of fish. The enzyme was stable at pH 6 to 9 in the presence of calcium ions. Km and kcat values of the enzyme for N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester were quite similar to those of catfish anionic and bovine cationic trypsins.     

Detergent Formulations for Wool Domestic Washings Containing Immobilized Enzymes

The stability of immobilized and native Esperase, a commercial serine protease, was studied by incubating the enzymes in four formulations containing the same amount of anionic and non-ionic surfactants. The results show that the activity of the immobilized enzyme is not affected by the presence of detergents while the native enzyme lost 50% of activity after 20 min of incubation in these four formulations. The washing performance of the detergents prepared with the immobilized Esperase was studied on cotton and wool fabric samples stained with human blood and egg yolk, using as control the detergent containing native Esperase. The best stain removal for cotton samples stained with human blood was achieved using the detergent with immobilized Esperase. Several physical tests confirmed that wool keratin was not degraded by the immobilized Esperase, validating the ability to use formulated detergents containing this immobilized enzyme for safe wool domestic washing.     

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site.     

Pancreatic anionic trypsin: evidence for the existence of a 30 kDa form.

1. An anionic form of trypsin has been isolated from pancreas of various species (rat, pig, dog and cow). 2. The purification procedure included affinity chromatography on STI-Sepharose 4B and ion-exchange chromatography on DEAE-Sephadex A-50. 3. The preparation was homogenous as checked by SDS-polyacrylamide slab gel electrophoresis, resulting in an estimated molecular weight of 30 kilodaltons (kDa) for this anionic form. 4. Antibodies against the anionic form from rat pancreas cross-reacted towards the anionic enzyme from porcine pancreas but not with the dog or bovine enzyme, nor with all the studied cationic forms. 5. Limited proteolysis of tubulin, a cytoskeletal protein, with an anionic or cationic form of trypsin showed striking differences in the size of produced peptides.     

The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.     

Human glutathione S-transferases. Characterization of the anionic forms from lung and placenta.

Anionic glutathione S-transferases were purified from human lung and placenta. Chemical and immunochemical characterization, including polyacrylamide-gel electrophoresis, gave strong evidence that the anionic lung and placental enzymes are chemically similar, if not identical, proteins. The electrophoretic mobilities of both proteins were identical in conventional alkaline gels as well as in gels containing sodium dodecyl sulphate. Gel filtration of the intact active enzyme established an Mr value of 45000; however, with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under dissociating conditions a subunit Mr of 22500 was obtained. Amino acid sequence analysis of the N-terminal region of the placental enzyme revealed a single polypeptide sequence identical with that of lung. Results obtained from immunoelectrophoresis, immunotitration, double immunodiffusion and rocket immunoelectrophoresis also indicated the anionic lung and placental enzymes to be closely similar. The chemical similarity of these two proteins was further supported by protein compositional analysis and fragment analysis after chemical hydrolysis. Immunochemical comparison of the anionic lung and placental enzymes with human liver glutathione S-transferases revealed cross-reactivity with the anionic omega enzyme, but no cross-reactivity was detectable with the cationic enzymes. Comparison of the N-terminal region of the human anionic enzyme with reported sequences of rat liver glutathione S-transferases gave strong evidence of chemical similarity, indicating that these enzymes are evolutionarily related. However, computer analysis of the 30-residue N-terminal sequence did not show any significant chemical similarity to any other reported protein sequence, pointing to the fact that the glutathione S-transferases represent a unique class of proteins.     

The antibacterial properties of secreted phospholipases A(2)

There is a considerable body of evidence to support the antibacterial properties of the group IIa phospholipase A(2) as an important physiological function. This enzyme is able to act as an acute phase protein and may be part of the innate defence system of the body, acting in concert with other antibacterial proteins and peptides. The enzyme is most effective against Gram-positive bacteria whereas penetration of the lipopolysaccharide coat of Gram-negative bacteria requires bactericidal/permeability-increasing protein (BPI) as an additional permeabilizing factor. The global cationic nature of this protein (pI>10.5) appears to facilitate penetration of the anionic bacterial cell wall. In addition, the considerable preference of the enzyme for anionic phospholipid interfaces provides specificity toward anionic bacterial membranes as opposed to zwitterionic eucaryotic cell membranes.     

Comparison of salt effects on the reactions of acetylcholinesterase with cationic and anionic inhibitors

The influence of inorganic salts on the inhibition of acetylcholinesterase by charged organophosphorous inhibitors has been studied. It has been shown that the salt effect on the reaction of acetylcholinesterase with anionic bis(p-nitrophenyl) phosphate is determined by the influence of added salts on the activity coefficient of the inhibitor. In contrast to the salt effects on the reaction of acetylcholinesterase with cationic compounds, it does not include contribution from the enzyme charges. The smaller salt effect in the case of anionic inhibitor can be explained assuming that the anionic inhibitor does not form a non-covalent complex with the enzyme before the phosphorylation step of the reaction. Comparison of salt effects on the substrate turnover showed that in the case of cholinesterases from natural sources they are larger than in the case of enzymes expressed in recombinant cell clones. The enhanced salt effects may result from post-translational modification of the enzyme.     

Blood group activity of human sucrase from intestinal metaplasia.

Sucrases were purified from human small intestine and from areas of intestinal metaplasia of the stomach mucosa surrounding stomach cancers. The kinetic constants and pH activity profiles of enzyme preparations from the two sources were similar. No blood group activity of sucrase was detectable in preparations from three cases of intestinal metaplasia, but preparations from two other cases showed activity like that of the small intestine. These results indicate that sucrase from areas of intestinal metaplasia has similar enzymatic properties to those of enzyme from the small intestine, but that the antigenic sugar moiety of the enzyme associated with blood group activity varies.     

Effects of surfactant mixtures, including Corexit 9527, on bacterial oxidation of acetate and alkanes in crude oil

Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil by Acinetobacter calcoaceticus ATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation by A. calcoaceticus ATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed for A. calcoaceticus ATCC 31012.     

Effects of surfactants on the activity of phospholipase D.

We studied the dependence of the activity of cabbage phospholipase A on the substrate (phosphatidylcholine) the aggregated state of which is regulated by addition of either anionic (sodium dodecyl sulfate, cholate or oleate) or cationic (cetyl-trimethylammonium bromide) surfactants. Activation of the enzyme induced by anionic surfactants was shown to correlate with the size of their polar groups. The phospholipase hydrolase activity correlated with the transformation of multilayer liposomes into micelles. The relationship between the processes was of a complex character. The dependence of the amount of enzymically released choline on the calcium concentration passed through a sharp maximum in the presence of the anionic detergents and monotonically increased in the presence of the cationic detergent. In the former case, the sharp increase in the enzyme activity was suggested to be caused by precipitation of phospholipase D with the anionic detergent calcium salt, which can be considered as a specific type of immobilization.     

Amyloid-cholinesterase interactions. Implications for Alzheimer's disease

Acetylcholinesterase is an enzyme associated with senile plaques. Biochemical studies have indicated that acetylcholinesterase induces amyloid fibril formation by interaction throughout the peripherical anionic site of the enzyme forming highly toxic acetylcholinesterase-amyloid-beta peptide (Abeta) complexes. The pro-aggregating acetylcholinesterase effect is associated with the intrinsic amyloidogenic properties of the corresponding Abeta peptide. The neurotoxicity induced by acetylcholinesterase-Abeta complexes is higher than the that induced by the Abeta peptide alone, both in vitro and in vivo. The fact that acetylcholinesterase accelerates amyloid formation and the effect is sensitive to peripherical anionic site blockers of the enzyme, suggests that specific and new acetylcholinesterase inhibitors may well provide an attractive possibility for treating Alzheimer's disease. Recent studies also indicate that acetylcholinesterase induces the aggregation of prion protein with a similar dependence on the peripherical anionic site.     

A new flavin radical signal in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae. An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.     

Multiple factors influence the binding of a soluble, Ca2+-independent, diacylglycerol kinase to unilamellar phosphoglyceride vesicles

We studied the influence of membrane lipids, MgCl2, and ATP on the ability of a soluble diacylglycerol kinase to bind to 100-nm lipid vesicles. The enzyme did not bind detectably to vesicles that contained phosphatidylcholine alone or to vesicles that contained 50 mol % phosphatidylcholine + 50 mol % phosphatidylethanolamine. But it did bind to vesicles that contained anionic phosphoglycerides, and maximal binding occurred (in the presence of MgCl2) when the vesicles contained anionic phosphoglycerides alone. When increasing amounts of phosphatidylcholine were included in phosphatidylserine-containing vesicles, enzyme binding to the vesicles decreased by as much as 1000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in binding occurred until the concentration of phosphatidylserine was reduced to below 25 mol %. These results and results obtained with vesicles that contained various mixtures of anionic phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, and unesterified cholesterol provided evidence that anionic phosphoglycerides were positive effectors of binding, phosphatidylcholine was a negative effector, and phosphatidylethanolamine and unesterified cholesterol were essentially neutral diluents. Other experiments showed that diacylglycerol and some of its structural analogues also were important, positive effectors of enzyme binding and that addition of ATP to the medium increased their effects. The combined results of the study suggest that the enzyme may bind to vesicles via at least two types of binding sites: one type that requires anionic phospholipids and is enhanced by Mg2+ but inhibited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.