Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.     

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.     

On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration

Monitoring CO2 production in systems, where pH is changing with time is hampered by the chemical behavior and pH-dependent volatility of this compound. In this article, we present the first method where the concentration and production rate of dissolved CO2 can be monitored directly, continuously, and quantitatively under conditions where pH changes rapidly ( approximately 2 units in 15 min). The method corrects membrane inlet mass spectrometry (MIMS) measurements of CO2 for pH dependency using on-line pH analysis and an experimentally established calibration model. It is valid within the pH range of 3.5 to 7, despite pH-dependent calibration constants that vary in a non-linear fashion with more than a factor of 3 in this interval. The method made it possible to determine the carbon dioxide production during Lactococcus lactis fermentations, where pH drops up to 3 units during the fermentation. The accuracy was approximately 5%. We used the method to investigate the effect of initial extracellular pH on carbon dioxide production during anarobic glucose fermentation by non-growing Lactocoocus lactis and demonstrated that the carbon dioxide production rate increases considerably, when the initial pH was increased from 6 to 6.8.     

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.     

Cardiac-specific Nkx2.5 homeodomain: conformational stability and specific DNA binding of Nkx2.5(C56S)

The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound to the same specific DNA. The thermal unfolding of Nkx2.5(C56S) at pH 6.0 or 7.4 is a reversible, two-state process with unit cooperativity, as measured by differential scanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) at pH 7.4 increases T(m) from 44 to 54 +/- 0.2 degrees C and DeltaH from 34 to 45 +/- 2 kcal/mol (giving a DeltaC(p) of approximately 1.2 kcal K(-)(1) mol(-)(1) for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at <37 degrees C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2-6 x 10(8) M(-)(1) from 10 to 37 degrees C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of DeltaH gives a DeltaC(p) of -0.18 +/- 0.01 kcal K(-)(1) mol(-)(1). This corresponds to 648 +/- 36 A(2) of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Acyl-coenzyme A binding protein expression alters liver fatty acyl-coenzyme A metabolism

Although studies in vitro and in yeast suggest that acyl-CoA binding protein ACBP may modulate long-chain fatty acyl-CoA (LCFA-CoA) distribution, its physiological function in mammals is unresolved. To address this issue, the effect of ACBP on liver LCFA-CoA pool size, acyl chain composition, distribution, and transacylation into more complex lipids was examined in transgenic mice expressing a higher level of ACBP. While ACBP transgenic mice did not exhibit altered body or liver weight, liver LCFA-CoA pool size increased by 69%, preferentially in saturated and polyunsaturated, but not monounsaturated, LCFA-CoAs. Intracellular LCFA-CoA distribution was also altered such that the ratio of LCFA-CoA content in (membranes, organelles)/cytosol increased 2.7-fold, especially in microsomes but not mitochondria. The increased distribution of specific LCFA-CoAs to the membrane/organelle and microsomal fractions followed the same order as the relative LCFA-CoA binding affinity exhibited by murine recombinant ACBP: saturated > monounsaturated > polyunsaturated C14-C22 LCFA-CoAs. Consistent with the altered microsomal LCFA-CoA level and distribution, enzymatic activity of liver microsomal glycerol-3-phosphate acyltransferase (GPAT) increased 4-fold, liver mass of phospholipid and triacylglyceride increased nearly 2-fold, and relative content of monounsaturated C18:1 fatty acid increased 44% in liver phospholipids. These effects were not due to the ACBP transgene altering the protein levels of liver microsomal acyltransferase enzymes such as GPAT, lysophosphatidic acid acyltransferase (LAT), or acyl-CoA cholesterol acyltransferase 2 (ACAT-2). Thus, these data show for the first time in a physiological context that ACBP expression may play a role in LCFA-CoA metabolism.