Influence of "energization" on the binding of M protein with p-nitrophenyl alpha-D-galactopyranoside.
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A specific binding of p-nitrophenyl alpha-D-galactopyranoside has been measured by flow dialysis with Escherichia coli ML 308225 membrane vesicles containing the lac carrier protein. The number of binding sites, 0.45 nmol/mg of membrane protein, remains unchanged in the presence or absence of energy. On the other hand, "energization" increases the M protein affinity for p-nitrophenyl alpha-D-galactopyranoside. The dissociation constant (Kd) is 4 and 21 microM in the presence and absence, respectively, of D-lactate. The same energization effects are found with E. coli A3245 membrane vesicles. p-Nitrophenyl alpha-D-galactopyranoside can be used as a substrate to study energization effect on binding to the lactose permease M protein. These results corroborate observations that energy increases the lac carrier protein affinity for its substrate, and they also confirm the concentration of the M protein, which corresponds to 1.4% of the membrane protein. (+info)
Dependence on pH of parameters of lactose transport in Escherichia coli. Evidence for an essential protonated group of the carrier.
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The kinetic parameters Km and V of transported by the lactose permease of Escherichia coli have been explored in the pH range 4.8--9.2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o-nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton-motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation. Ktransport for methylthiogalactoside and for o-nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4.8 and 8.0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8--8.4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply. The role of energization in substrate-carrier binding and the role of the essential protonation in the context of the proton symptom model are discussed. (+info)
Discrimination between activity of (alpha 2-3)-sialyltransferase and (alpha 2-6)-sialyltransferase in human platelets using p-nitrophenyl-beta-D-galactoside as acceptor.
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Exogenous asialo-glycoproteins and endogenous acceptors are both sialylated by incubating cytidine 5'-monophosphate N-[14C]acetylneuraminic acid (CMP [14C]NeuAc) with a lysate of human platelets but their respective incorporation levels vary with the divalent cation concentration. P-Nitrophenyl-beta-D-galactoside has also been demonstrated to be an acceptor of sialyl residues, and two different sialyl derivatives are synthesized according to the concentration of divalent cations. P-Nitrophenyl-beta-D-[6-3H]galactoside has been prepared by reduction with tritiated borohydride of the compound previously oxidized by galactose oxidase. Using this labelled p-nitrophenyl-beta-D-galactoside as acceptor and unlabelled CMP-NeuAc as donor, the two sialyl derivatives have been identified by methylation analysis as alpha-sialosyl-(2-3)-p-nitrophenyl-beta-D-galactoside and alpha-sialosyl-(2-6)-p-nitrophenyl-beta-D-galactoside. In addition to their different responses to divalent cation requirements, the sialyltransferase activities responsible for the synthesis of the two sialylgalactoside isomers have been clearly distinguished by their temperature and pH optimal values. They also exhibit different susceptibilities to dithioerythritol and different stabilities. These results demonstrate the presence in human platelets of two sialyltransferases: a CMP-NeuAc: galactoside (alpha 2-3)-sialyltransferase and a CMP-NeuAc: galactoside (alpha 2-6)-sialyltransferase. (+info)
Catalysis by the large subunit of the second beta-galactosidase of Escherichia coli in the absence of the small subunit.
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Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated. The kcat. values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The kcat. values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates. Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol. Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga. beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes. Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change. Values of kcat. for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar. The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance. (+info)
Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling with synthetic inhibitors.
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Kinetic parameters for the inactivation of the 6-phospho-beta-galactosidase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of 2,4-dinitrophenyl-2-deoxy-2-halogeno- galactoside-6-phosphates have been determined. These inhibitors function by the formation of a stabilised glycosyl-enzyme intermediate. Inactivation and reactivation studies indicate that the fluoro derivative is formed most rapidly, but is also hydrolysed fastest. The chloro derivative forms the most stable covalent intermediate. HPLC profiles of V8-protease digestion of native and inhibited protein show significant differences, whereas the inhibited 6-phospho-beta-galactosidase and a point mutant of 6-phospho-beta- galactosidase (E375Q) yield the same proteolytic fragments. The suggestion that E375 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two peptides, residues 336-375 and 376-383, are not produced, due to the absence of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of the respective V8 cleavage site due to the chemical reaction of the inhibitor at position 375. Specific modification of the glycosyl bond between the inhibitor and E375 by aminolysis with benzylamine generated a glutamatic-acid-5-benzylamide complex at that position in the peptide. The Edman derivative of the modified E375 appears to be stable and was isolated by Edman degradation of trypsin-digested V8-peptide. It was shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-galactosidase, consistent with previous findings for enzymes in this family. (+info)
Inactivation of membrane transport in Escherichia coli by near-ultraviolet light.
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Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways. It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane. Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves. In agreement, we found that the active accumulation of [14C]thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system. The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport. As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells. Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates. With the use of such cells, it was found that high fluences (doses) made the cells leaky. Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented. It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system. In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol. (+info)
Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product.
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The nucleotides of the xynB gene of Clostridium stercorarium F-9 were sequenced. The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377. The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus. Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E. coli and C. stercorarium F-9. The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside. The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C. This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min. (+info)
Energy cost of galactoside transport to Escherichia coli.
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Energy reserves of Escherichia coli can be depleted by our previously reported procedure to a level such that even the "downhill" transport of o-nitrophenyl-beta-D-galactopyranoside (ONPG) is completely dependent upon the exogenous energy supply. The ONPG concentration is high externally to the cells and is low intracellular because of the action of cytoplasmic beta-galactosidase. In the present work, depleted cell suspensions have been infused at low, steady rates with glucose and other energy sources while measurements of transport were being made. Comparing the rate of ONPG transport with the rate of introduction of glucose under conditions where the chosen glucose infusion rate limits transport, we find that 89 molecules of ONPG are transported per molecule of fully oxidized glucose. This transport yield is constant over a 6.5-fold range in rate of glucose addition. This constancy over a range of infusion rates implies that transport is the major cellular function under these special conditions. The yield value if 89 is in the agreement with the predicitions of 76 from Mitchell's chemiosmotic theory and constitutes an independent proff of its validity, since all the other proposed mechanisms of engery coupling predict much smaller yields. The lag from the start of glucose infusion into the reaction cuvette, to the extrapolated time at which a steady rate of transport and concomitant hydrolysis are achieved, is short (approximately 1 min). Similarly, the time after the infusion is stopped until the rate of transport returns to the background rate is also short. The latter implies that the energy metabolism is directed almost entirely to transport and/or other ongoing cellular processes and not to repair or renewal of an energy-independent, facilitated diffusion system. (+info)