Mutation E252C Increases Drastically theKmValue for Na+and Causes an Alkaline Shift of the pH Dependence of NhaA Na+/H+Antiporter ofEscherichia coli

SummaryA re-examination of the affinity of pro-urokinase (pro-UK), HMW and LMW-urokinase (UK) to fibrin/Celite was undertaken in order to explain how the chance purification of pro-UK from freshly voided urine by fibrin/Celite affinity chromatography may be reconciled with the subsequent observations that pro-UK failed to bind significantly to fibrin clots in plasma. A significant pH dependence of pro-UK binding to fibrin/Celite was found. Substantial binding of pro-UK (native or recombinant from E. coli), but not of the two-chains forms, was seen at about pH 6.5, which is in the normal pH range of pooled, freshly voided urine. By contrast, at pH 7.4 fibrin binding of pro-UK was much reduced, though it was still significantly greater than that of HMW or LMW-UK. This finding helps to explain the fibrin-binding of pro-UK in freshly voided urine but not in blood. In order to determine if this' pH dependence was the sole explanation for why pro-UK could not be isolated by this method from stored urine, the stability of pro-UK in urine was evaluated by incubating 125I-labeled pro-UK in urine: Incubation for up to 4 days (37° C) was not accompanied by any degradation of the single-chain pro-UK as evidenced by autoradiography under reducing conditions. It was concluded that the alkaline shift in pH which occurs in urine left standing, rather than the degradation of pro-UK, explained why freshly voided urine was found to be essential. Clot lysis studies at pH 6.5 and 7.4 showed no promotion of fibrinolysis at the pH which favored fibrin/Celite binding. Therefore, while the present study defines the conditions under which pro-UK may be purified from urine by fibrin/Celite chromatography, it provides no evidence that this binding phenomenon plays any role in fibrinolysis.

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pH dependence of the Slc4a11-mediated H+ conductance is influenced by intracellular lysine residues and modified by disease-linked mutations

AJP Cell Physiology ◽

10.1152/ajpcell.00128.2020 ◽

2020 ◽

Vol 319 (2) ◽

pp. C359-C370 ◽

Cited By ~ 2

Author(s):

Bianca N. Quade ◽

Aniko Marshall ◽

Mark D. Parker

Keyword(s):

Ph Dependence ◽

Corneal Dystrophy ◽

Corneal Endothelial Cells ◽

Wild Type ◽

Intracellular Loop ◽

K Value ◽

Alkaline Shift ◽

Lysine Residues ◽

The Hill ◽

Transmembrane Span

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance ( Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a p K value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in p K. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in p K, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in p K. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.

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A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm ofEscherichia coli

Protein Expression and Purification ◽

10.1016/s1046-5928(05)80026-9 ◽

1992 ◽

Vol 3 (2) ◽

pp. 108-113

Author(s):

H ENGEL ◽

H MOTTL ◽

W KECK

Keyword(s):

Fusion Proteins ◽

Protein A ◽

Ofescherichia Coli ◽

Staphylococcal Protein A ◽

Staphylococcal Protein ◽

High Level

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Self-association of insulin. Its pH dependence and effect of plasma

Diabetes ◽

10.2337/diabetes.36.3.261 ◽

1987 ◽

Vol 36 (3) ◽

pp. 261-264 ◽

Cited By ~ 15

Author(s):

E. Helmerhorst ◽

G. B. Stokes

Keyword(s):

Ph Dependence ◽

Self Association

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Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

10.26434/chemrxiv.7985618.v1 ◽

2019 ◽

Author(s):

Adrian Roitberg ◽

Pancham Lal Gupta

Keyword(s):

Transfer Reaction ◽

Catalytic Mechanism ◽

Ph Dependence ◽

Ph Effects ◽

Dependent Manner ◽

E Coli ◽

Gar Tfase ◽

Activity Curve ◽

Ph Dependent ◽

Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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pH Dependence of the polarographic limiting currents of α-keto acids

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19610141 ◽

1961 ◽

Vol 26 (1) ◽

pp. 141-155 ◽

Cited By ~ 35

Author(s):

S. Ono ◽

M. Takagi ◽

T. Wasa

Keyword(s):

Ph Dependence ◽

Keto Acids

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Photoreaction of 3,4-dimethoxy-1-nitrobenzene with butylamine. A pH dependence of regioselectivity

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19861665 ◽

1986 ◽

Vol 51 (8) ◽

pp. 1665-1670 ◽

Cited By ~ 6

Author(s):

Petr Kuzmič ◽

Libuše Pavlíčková ◽

Jiří Velek ◽

Milan Souček

Keyword(s):

Ph Dependence ◽

Molar Ratio

Irradiation of 3,4-dimethoxy-1-nitrobenzene in the presence of butylamine leads to the formation of both possible photosubstitution products, i.e., 2-methoxy-4-nitro-N-butylaniline and 2-methoxy-5-nitro-N-butylaniline with the predominance of the latter. Regioselectivity of the reaction as measured by molar ratio of the two isomeric products varies with pH of the solution, ranging from 3 : 1 at pH 10 to 12. The results are discussed in view of possible use of 3,4-dimethoxy-1-nitrobenzene moiety as a lysine-directed photoaffinity probe.

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Electroanalytical chemistry of vanadium complexes: Electrochemical oxidation of [V(IV)O(nta)(H2O)]-

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19890064 ◽

1989 ◽

Vol 54 (1) ◽

pp. 64-69 ◽

Cited By ~ 4

Author(s):

Roland Meier ◽

Gerhard Werner ◽

Matthias Otto

Keyword(s):

Cyclic Voltammetry ◽

Electrochemical Oxidation ◽

Ph Dependence ◽

Differential Pulse ◽

Nitrilotriacetic Acid ◽

Reduced Form ◽

Vanadium Complexes ◽

Differential Pulse Polarography ◽

Electroanalytical Chemistry ◽

Ph Range

Electrochemical oxidation of [V(IV)O(nta)(H2O)]- (H3nta nitrilotriacetic acid) was studied in aqueous solution by means of cyclic voltammetry, differential pulse polarography, and current sampled DC polarography on mercury as electrode material. In the pH-range under study (5.5-9.0) the corresponding V(V) complex is produced by one-electron oxidation of the parent V(IV) species. The oxidation product is stable within the time scale of cyclic voltammetry. The evaluation of the pH-dependence of the half-wave potentials leads to a pKa value for [V(IV)O(nta)(H2O)]- which is in a good agreement with previous determinations. The measured value for E1/2 is very close to the formal potential E0 calculated via the Nernst equation on the basis of known literature values for log Kox and log Kred, the complex stability constants for the oxidized and reduced form, respectively.

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