Effects of perfusate buffer capacity on capillary CO2-HCO3(-)-H+ reactions: theory.

The importance of perfusate nonbicarbonate buffer capacity (beta nonHCO3) to intracapillary CO2-HCO3(-)-H+ reactions was assessed by theoretical analysis of CO2 exchange in saline-perfused pulmonary capillaries. Time courses for perfusate PCO2, [HCO3-], and [H+] were computed for capillaries containing different activities of luminal vascular carbonic anhydrase and different amounts of perfusate nonbicarbonate buffers. Mobilization of perfusate HCO3- toward CO2 during capillary transit is determined by the availability of HCO3- and H+. A supply of protons from the nonbicarbonate buffer pool is necessary to maintain a high rate of HCO3- dehydration. The analyses indicate that beta nonHCO3 has marked nonlinear effects on transcapillary CO2 exchange and intravascular pH equilibration. These nonlinear effects differ from those previously computed for CO2 reactions in an open system because the present model system consists of a sequential combination of open (within capillary proper) and closed (within postcapillary vasculature) systems. The role of luminal vascular carbonic anhydrase in capillary CO2 reactions is strongly dependent on beta nonHCO3. Perfusate nonbicarbonate buffer capacity must be considered when the results of experimental studies of transcapillary CO2 exchange and/or intravascular pH equilibration are interpreted.     

On calculating concentrations of "HCO3" from pH and PCO2

1. As used in the Henderson-Hasselbalch equation, [HCO3], [CO2] and pH may all be variously defined; values of pK'1 must be chosen accordingly. 2. In common usage, "HCO3" may include CO3, carbamate, various ion pairs and possibly other bound CO2, as well as free HCO3 ions. 3. pH measurements may be systematically affected by the choice of standard buffers and by proteins and blood cells, and the errors in pH may be pH-dependent. 4. According to how it is expressed, the solubility coefficient for CO2 (S) may be influenced by sample water content, proteins and lipids. However, it need not feature in the calculation. 5. pK'1 is often found to decrease with increasing pH. This may be partly due to inclusion of CO3 and carbamate, but not of H2CO3.HCO3-, in "HCO3" and partly, perhaps, to errors in pH measurement. 6. To the extent that pH measurements are reliable, concentrations or activities of true HCO3 are calculable from pH and PCO2, but, if pH measurements are likely to be systematically erroneous, it may be preferable to define "HCO3" as "total bound CO2" and to base pK'1 on gasometric or titrimetric determinations of that.     

Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

The role of CO2 in cobalt-catalyzed peroxidations

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably due to its sequestration of the Co(II). When CO(2) was used, there was an initial burst of NADH oxidation followed by a slower linear rate. The presence of carbonic anhydrase eliminated this initial burst; establishing that CO(2) rather than HCO(3)(-) was the species responsible for the observed rate enhancements. Both kinetic and spectral data indicated that Co(II) was converted by H(2)O(2) into a less active form from which Co(II) could be regenerated. This less active form absorbed in both the UV and visible regions, and is assumed to be a peroxy bridged binuclear complex. The rate of formation of this absorbing form was increased by HCO(3)(-)/CO(2). A minimal mechanism consistent with these observations is proposed.     

Differential respiratory effects of HCO3- and CO2 applied on ventral medullary surface of rats

To estimate whether H+ is the unique stimulus of the medullary chemosensor, ventilatory effects of HCO3- and/or CO2 applied on the ventral medullary surface using an improved superfusion technique and of CO2 inhalation were compared in halothane-anesthetized spontaneously breathing rats. Superfusion with low [HCO3-]-acid mock cerebrospinal fluid (CSF) (normal Pco2) induced a significant increase in ventilation, with an accompanying reduction in endtidal Pco2 (PETco2). High [HCO3-]-alkaline CSF depressed ventilation. Changes in Pco2 of superfusing CSF, on the other hand, had no significant effect despite the similar changes in pH. Simultaneous decrease in [HCO3-] and Pco2 of mock CSF with normal pH also maintained stimulated respiration. CO2 inhalation during superfusion with various [HCO3-] solutions caused further increase in ventilation as PETco2 increased. The results suggest that the surface area of the rat ventral medulla contains HCO3- (or H+)-sensitive respiratory neural substrates which are, however, little affected by CO2 in the subarachnoid fluid. A CO2 (or CO2-induced H+)-sensitive chemosensor responsible for the increase in ventilation during CO2 inhalation may exist elsewhere functionally apart from the HCO3- (or H+)-sensitive sensor in the examined surface area.     

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.     

Control of active proton transport in turtle urinary bladder by cell pH

The rate of active H+ secretion (JH) across the luminal cell membrane of the turtle bladder decreases linearly with the chemical (delta pH) or electrical potential gradient (delta psi) against which secretion occurs. To examine the control of JH from the cell side of the pump, acid-base changes were imposed on the cellular compartment by increasing serosal[HCO3-] at constant PCO2 or by varying PCO2 at constant [HCO3-]. When serosal [HCO3-] was increased from 0 to 60 mM, cell [H+] decreased, as estimated by the 5,5-dimethyloxazoladine-2,4- dione method. JH was a saturable function of cell [H+], with an apparent Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal [HCO3-], the PCO2 required to reach a maximal JH increased with [HCO3-] so that JH was a function of cell [H+] rather than of cell [HCO3-] or CO2. The proton pump was controlled asymmetrically with respect to the pH component of the electrochemical potential for protons, microH. On the cell side of the pump, a delta pH of < 1 U was required to vary JH between maximal and zero values, whereas on the luminal side a delta pH of 3 U was required. Cell [H+] regulates JH by determining the availability of H+ to the pump in a relationship resembling Michaelis-Menten kinetics. Increasing luminal [H+] generates an energy barrier at a luminal pH near 4.4 that equals the free energy (per H+ translocated) of the metabolic driving reaction.     

Na(+)-dependent Cl-HCO3 exchange in the squid axon. Dependence on extracellular pH.

Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths

Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (～10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.     

pH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.     

Carbon dioxide storage and nonbicarbonate buffering in the human body before and after an Himalayan expedition

Before and 7-12 days after an Himalayan expedition CO2 equilibration curves were determined in the blood plasma of 12 mountaineers by in vitro and in vivo CO2 titration; in vivo osmolality changes (delta Osm x deltaPCO2(-1), deltaOsm x delta pH(-1), where PCO2 is the partial pressure of CO2) during the latter experiments yielded estimates of whole body CO2 storage. In vitro -delta[HCO3-] x delta pH(-1) [nonbicarbonate buffer capacity (beta) of blood] was increased 7 days after descent [before 31.3 (SEM 0.4) mmol x kgH2O(-1), after 38.3 (SEM 3.9) mmol x kgH2O(-1); P<0.05] resulting from an increased proportion of young erythrocytes; in additional experiments an augmented beta was found in young (low density cells) compared to old cells [<1.097 g x ml(-1): 0.216 (SEM 0.028) mmol x gHb(-1), >1.100 g x ml(-1): 0.145 (SEM 0.013) mmol x gHb(-1), where Hb is haemoglobin; P < 0.02]. In spite of increased Hb mass in vivo delta[CO2total] x deltaPCO2(-1) [0.192 (SEM 0.010) mmol x kgH2O(-1) x mmHg(-1)] and -delta[HCO3-] x delta pH(-1) [17.9 (SEM 1.0) mmol x kgH2O(-1)] as indicators of extracellular beta rose only slightly after altitude (7 days +16%, P<0.02; +7%, NS) because of haemodilution. The deltaOsm x deltaPCO2(-1) [0.230 (SEM 0.015) mosmol x kgH2O(-1) x mmHg(-1)] remained unchanged. Prealtitude differences in deltaOsm x delta pH(-1) between hypercapnia [-41 (SEM 5) mosmol x kgH2O(-1)] and hypocapnia [-20 (SEM 3) mosmol x kgH2O(-1); P<0.01] disappeared temporarily after return since the former slope was reduced. The high value during hypercapnia before ascent probably resulted from mechanisms stabilizing intracellular pH during moderate hypercapnia which were attenuated after descent.     

Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic anhydrase, bicarbonate, and unstirred layers

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Bicarbonate and other buffer systems can enhance the rate of H+ diffusion through mucus in vitro.

The effect of various diffusible buffers on mucus H+ permeability, and in particular the potency of the HCO3-/CO2 buffer system relative to other selected buffers is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layer was most pronounced for low DH+ values near neutrality. This concentration dependence was most marked with mucus layers in the buffer systems investigated. Furthermore, the nature and pKa values of the diffusible buffer systems used in this study had a profound effect on measured DH+. The effect was particularly striking in the case of HCO3- buffer with mucus. Possible implications of these in vitro findings in mucosal protection from acid are discussed.     

Determinants of Deoxyglucose Uptake in Cultured Astrocytes: The Role of the Sodium Pump

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.     

Bicarbonate-dependent peroxidase activity of human Cu,Zn-superoxide dismutase induces covalent aggregation of protein: intermediacy of tryptophan-derived oxidation products

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3-did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3., forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.     

Mechanism of hydrogen peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations

We have reinvestigated the biochemistry of H2O2-induced Cu,Zn-superoxide dismutase (SOD1)-centered radicals, detecting them by immuno-spin trapping. These radicals are involved in H2O2-induced structural and functional damage to SOD1, and their mechanism of generation depends on copper and/or (bi)carbonate (i.e., CO2, CO3(-2), or HCO3-). First, in the absence of DTPA and (bi)carbonate, Cu(II) was partially released and rebound at His, Cys, and Tyr residues in SOD1 with the generation of protein-copper-bound oxidants outside the SOD1 active site by reaction with excess H2O2. These species produced immuno-spin trapping-detectable SOD1-centered radicals associated with H2O2-induced active site ( approximately 5 and approximately 10 kDa fragments) and non-active site (smearing between 3 and 16 kDa) copper-dependent backbone oxidations and subsequent fragmentation of SOD1. Second, in the presence of DTPA, which inhibits H2O2-induced SOD1 non-active site fragmentation, (bi)carbonate scavenged the enzyme-bound oxidant at the SOD1 active site to produce the carbonate radical anion, CO3*-, thus protecting against active site SOD1 fragmentation. CO3*- diffuses and produces side chain oxidations forming DMPO-trappable radical sites outside the enzyme active site. Both mechanisms for generating immuno-spin trapping-detectable SOD1-centered radicals were susceptible to inhibition by cyanide and enhanced at high pH values. In addition, (bi)carbonate enhanced H2O2-induced SOD1 turnover as demonstrated by an enhancement in oxygen evolution and SOD1 inactivation. These results help clarify the free radical chemistry involved in the functional and structural oxidative damage to SOD1 by H2O2 with the intermediacy of copper- and CO3*--mediated oxidations.