Exclusion of Induced Bacteriophage from Cells of a Lysogenic Bacillus megaterium Committed to Sporulation

In Escherichia coli ML 308-225, d-ribose is transported into the cell by a constitutive active transport system of high activity. The activity of this transport system is severely reduced in cells subjected to osmotic shock, and the system is not present in membrane vesicles. The mechanism by which metabolic energy is coupled to transport of ribose was investigated. Substrates which generate adenosine 5'-triphosphate primarily through oxidative phosphorylation are poor energy sources for ribose uptake in DL-54, a mutant of ML 308-225 which lacks activity for the membrane-bound Ca(2+), Mg(2+)-dependent adenosine triphosphatase required for oxidative phosphorylation. Arsenate severely inhibits ribose uptake, whereas, under the same conditions, uptake of l-proline is relatively insensitive to arsenate. Anaerobiosis does not significantly inhibit ribose uptake in ML 308-225 or DL-54 when glucose is the energy source. A significant amount of ribose uptake is resistant to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol. These results indicate that the phosphate bond energy of adenosine 5'-triphosphate, rather than an energized membrane state, couples energy to ribose transport in ML 308-225.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

Mitochondrial calcium transport: mechanisms and functions

Ca(2+)transport across the mitochondrial inner membrane is facilitated by transporters having four distinct sets of characteristics as well as through the Ca(2+)-induced mitochondrial permeability transition pore (PTP). There are two modes of inward transport, referred to as the Ca(2+)uniporter and the rapid mode or RaM. There are also two distinct mechanisms mediating outward transport, which are not associated with the PTP, referred to as the Na(+)-dependent and the Na(+)-independent Ca(2+)efflux mechanisms. Several important functions have been proposed for these mechanisms, including control of the metabolic rate for cellular energy (ATP) production, modulation of the amplitude and shape of cytosolic Ca(2+)transients, and induction of apoptosis through release of cytochrome c from the mitochondrial inter membrane space into the cytosolic space.The goals of this review are to survey the literature describing the characteristics of the mechanisms of mitochondrial Ca(2+)transport and their proposed physiological functions, emphasizing the more recent contributions, and to consider how the observed characteristics of the mitochondrial Ca(2+)transport mechanisms affect our understanding of their functions.     

Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca(2+) entry is uncertain. To assess the Ca(2+) source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.     

Vanadyl and vanadate inhibit Ca2+ transport systems of the adipocyte plasma membrane and endoplasmic reticulum

Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.     

Mechanism of energy coupling for transport of deoxycytidine, uridine, uracil, adenine and hypoxanthine in Escherichia coli.

The transport processes for uridine, deoxycytidine, uracil, adenine and hypoxanthine require an energy source and are active under anaerobic or aerobic conditions. Inhibitory effects of cyanide, arsenate, carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol and N,N'-dicyclohexylcarbodiimide on the transport of uridine and deoxycytidine differ from the corresponding effects on the transport of uracil, adenine and hypoxanthine. The nature of these inhibitory effects supports the conclusion that uridine and deoxycytidine transport is energized either by electron transport or by ATP hydrolysis via (Ca2+ + Mg2+)-ATPase. The transport or uracil, adenine and hypoxanthine is dependent upon ATP or some high energy phosphate derivative of ATP, but is independent of (Ca2+ + Mg+)-ATPase and electron transport. Uptake of the ribose moiety of uridine by a mutant of Escherichia coli B, which lacks the transport system for uracil and intact uridine, is neither stimulated by energy sources nor inhibited by various inhibitors of energy metabolism under either aerobic or anaerobic conditions.     

The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p

Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions. In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain. This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis. An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments.     

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.     

Effects of polyamines on mitochondrial Ca(2+) transport

Mammalian mitochondria are able to enhance Ca(2+) accumulation in the presence of polyamines by activating the saturable systems of Ca(2+) inward transport and buffering extramitochondrial Ca(2+) concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca(2+) concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca(2+) transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S(1) binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca(2+) transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S(1) site. Important physiological functions are related to activation of Ca(2+) transport systems by polyamines and their interactions with the S(1) site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Uncoupled ATP hydrolysis and thermogenic activity of the sarcoplasmic reticulum Ca2+-ATPase: coupling effects of dimethyl sulfoxide and low temperature

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

Transport of vitamin B12 in Escherichia coli: energy dependence.

This paper presents some evidence that the osmotic shock-sensitive, energy-dependent transfer of vitamin B12 from outer membrane receptor sites into the interior of cells of Escherichia coli requires an energized inner membrane, without obligatory intermediation of adenosine 5'-triphosphate (ATP). The experiments measured the effects of glucose, D-lactate, anaerobiosis, arsenate, cyanide, and 2,4-dinitrophenol upon the rates of B12 transport by starved cells of E. coli KBT001, which possesses a functional Ca2+, Mg2+-stimulated adenosine triphosphatase (Ca,MgATPase), and of E. coli AN120, which lacks this enzyme. Both strains were able to utilize glucose and D-lactate aerobically to potentiate B12 transport, indicating that the Ca,MgATPase was not essential for this process. When respiratory electron transport was blocked, either by cyanide or by anaerobic conditions, and the primary source of energy for the cells was presumably ATP from glucose fermentation, the rate of B12 transport was much reduced in E. coli AN120 but not in E.coli KBT001. These results support the view that the CaMgATPase can play a role in B12 transport but only when the energy for this process must be derived from ATP. The results of experiments with arsenate also supported the conclusion that the generation of phosphate bond energy was not absolutely required for B12 transport.     

Correction by 1-25-dihydroxycholecalciferol of the abnormal fluidity and lipid composition of enterocyte brush border membranes in vitamin D-deprived rats

Weanling male Wistar rats were deprived of dietary and light sources of vitamin D for 11-18 weeks along with age-matched diet vitamin D-repleted controls to evaluate the role of lipid fluidity in the stimulatory effect of calcitriol on Ca transport. The "static" component of fluidity of proximal small intestine brush border membrane, as assessed by steady-state fluorescence techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene, was similar between these two groups. In contrast, the "dynamic" component of fluidity, as assessed by DL-2-(9-anthroyl)-stearic acid and DL-12-(9-anthroyl)-stearic acid, was decreased in membranes of D-deprived animals. Lipid composition was analyzed to evaluate the potential mechanism mediating these fluidity changes. In vitamin D-deprived rats, linoleic (18:2) and arachidonic (20:4) acids of the phosphatidylcholine and phosphatidylethanolamine fractions of the membrane were decreased, whereas palmitic (16:0) and stearic (18:0) acids were increased in the phosphatidylethanolamine fraction of the membrane. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between D-repleted and D-deprived rats. Membrane fluidity, lipid composition, and duodenal Ca transport were also analyzed 1, 2, and 5 h after the acute administration of 1-25-dihydroxycholecalciferol to D-deprived animals. In D-deprived rats, within 1-2 h, this hormone restored to levels of vitamin D-repleted controls the dynamic component of fluidity and concentrations of the same membrane phospholipid fatty acids. Since these changes temporally precede detectable increases in Ca absorption (demonstrable only during the 5th h), these data support the hypothesis that alterations in membrane fluidity and lipid composition may play an important role in the stimulation of intestinal calcium transport by calcitriol.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

HCO(3) Influx Across the Plasmalemma of Chara corallina: Divalent Cation Requirement

An absolute requirement for divalent cations is reported for H(14)CO(3) (-) influx in Chara corallina. Effective substitution of eluted Ca(2+) by Mg(2+) and Sr(2+) was observed, but Mn(2+) was completely ineffective in restoring H(14)CO(3) (-) transport activity. Similarly, La(3+) could not substitute for Ca(2+) in this system. Low concentrations of ethylenediaminetetraacetate (0.01 to 0.06 mm) significantly enhanced the rate at which H(14)CO(3) (-) transport capacity was lost.Examination of the response of OH(-) efflux, during Ca(2+)-free treatment, indicated that the cellular control over OH(-) efflux remained unaffected until membrane integrity became severely affected. This conclusion was supported by the response of OH(-) efflux to 10 mm K(+). Therefore, assimilation of H(14)CO(3) (-) is not rate-limited by an effect of Ca(2+) elution on the OH(-) transport system. Kinetic experiments indicated that Ca(2+) removal from the membrane resulted in noncompetitive inhibition of H(14)CO(3) (-) assimilation; the apparent Michaelis constant remained unaltered over a wide range of conditions. An hypothesis is presented which suggests that membrane integrity is necessary for HCO(3) (-) transport to occur, but Ca(2+) (Mg(2+), Sr(2+)), per se, must be bound to the transport complex before activity is established.