Functional reassembly of the coated vesicle proton pump

We have shown previously that treatment of the coated vesicle proton-translocating adenosine triphosphatase (H(+)-ATPase) with chaotropic agents results in the release of a set of peripheral polypeptides which includes the 73-, 58-, 40-, 34-, and 33-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973), with a coordinate loss of H(+)-ATPase activity. In the present paper we report the functional reassembly of the coated vesicle proton pump following dissociation of the peripheral subunits. Reassembly was demonstrated by restoration of ATP-driven proton transport using both native membranes and reconstituted vesicles and by Western blot analysis using a monoclonal antibody specific for the 73-kDa subunit. Reassembly occurs by attachment of a peripheral subcomplex containing the 73-, 58-, 34-, and 33-kDa subunits together with the 40-kDa polypeptide. The reassembled H(+)-ATPase, like the native proton pump, is inhibited by N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and N,N'-dicyclohexylcarbodiimide. Reassociation shows a biphasic time dependence, with restoration of 50-60% of the starting proton transport activity in the 1st h followed by recovery of a further 20-30% of the activity after 24 h. Reassembly also shows a marked dependence on protein concentration but, unlike solubilization of the intact H(+)-ATPase complex, does not require the presence of glycerol. Despite the ability of nucleotides to promote dissociation of the peripheral complex by chaotropic agents, reassociation is not blocked by the presence of 1 mM ATP. These results thus provide the first evidence for functional reassembly of a vacuolar H(+)-ATPase complex and should be useful in further analysis of the role of individual subunits in the assembly and activity of these ATP-driven proton pumps.     

Proteolysis and orientation on reconstitution of the coated vesicle proton pump

We recently proposed a structural model for the ATP-dependent proton pump from clathrin-coated vesicles (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802). To test this model further, we have carried out additional structural analysis of the (H+)-ATPase in both the detergent-solubilized and reconstituted states in this and the following paper (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). The orientation of the reconstituted proton pump was determined by analyzing the effect of detergent on ATP hydrolysis and by quantitating the extent of labeling of luminally oriented subunits using a membrane-impermeant reagent. Greater than 90% of the reconstituted (H+)-ATPase is oriented with the cytoplasmic surface facing outward. Treatment of the reconstituted (H+)-ATPase with trypsin results in rapid cleavage of the 100-, 73-, 58-, 38-, and 34-kDa subunits and slower cleavage of the 40- and 33-kDa subunits, consistent with our previous results indicating that all of these polypeptides have some portion of their mass exposed to the cytoplasmic surface. The 19- and 17-kDa subunits, by contrast, appear resistant to cleavage by trypsin in both the detergent-solubilized and reconstituted states, consistent with their being buried extensively in the hydrophobic phase of the bilayer. Treatment of the enzyme with trypsin under conditions in which the 100-, 73-, 58-, 38-, and 34-kDa subunits have been cleaved results in a species which is virtually inactive with respect to proton transport but retains 50% of the original ATPase activity, suggesting that proteolysis has resulted in uncoupling of these two activities. Cleavage of both the 73- and 58-kDa subunits by trypsin at a site 1-2 kDa from the amino terminus is inhibited in the presence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP, consistent with the suggestion that both the 73- and 58-kDa subunits may be nucleotide binding proteins.     

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.     

Structural characterization of the ATP-hydrolyzing portion of the coated vesicle proton pump

The ATP-hydrolyzing portion of the proton pump from clathrin-coated vesicles (isolated from calf brain) was solubilized with three nondenaturing detergents (cholate, octyl glucoside, and Triton X-100). The hydrodynamic properties of the solubilized (Mg2+)-ATPase were then determined by sedimentation analysis in H2O and D2O and gel filtration on Sepharose 4B. The coated vesicle (Mg2+)-ATPase migrated under all conditions as a single peak of activity. In cholate, the sedimentation coefficient (S20,w), Stokes radius (a), and partial specific volume (vc) were 8.25 (+/- 0.20) S, 68 (+/- 2) A, and 0.71 (+/- 0.03) cm3/g, respectively. In octyl glucoside and Triton X-100 these values were respectively 7.90 (+/- 0.20) and 7.45 (+/- 0.20) S, 68 (+/- 3) and 101 (+/- 5) A, and 0.74 (+/- 0.03) and 0.75 (+/- 0.03) cm3/g. Application of the Svedberg equation to these data gave a molecular weight for the protein-detergent complex of 217,000 +/- 21,000 (cholate), 234,000 +/- 26,000 (octyl glucoside), and 337,000 +/- 40,000 (Triton X-100). Assuming the protein binds one micelle of detergent, these values correspond to a protein molecular weight of 215,000 +/- 21,000 (cholate), 226,000 +/- 26,000 (octyl glucoside), and 247,000 +/- 40,000 (Triton X-100). The cholate-solubilized, gradient-purified (Mg2+)-ATPase, when combined with a 100,000 g pellet fraction, could be reconstituted by dialysis into phospholipid vesicles which displayed ATP-dependent proton uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [14C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump.     

The 40-kDa subunit enhances but is not required for activity of the coated vesicle proton pump.

We have previously demonstrated reassembly of a functional vacuolar (H+)-ATPase from clathrin-coated vesicles using the dissociated peripheral domain (V1) and the membrane-bound integral domain (V0) (Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836-14841). We have used this reassembly procedure to test the function of the 40-kDa subunit of the coated vesicle (H+)-ATPase. In the absence of V0, a fraction of the peripheral subunits reassemble into a V1 subcomplex which contains the 73-kDa A subunit, the 58-kDa B subunit, and the 34- and 33-kDa subunits but lacks the 40-kDa subunit. This subcomplex, which sediments with a mass of approximately 500 kDa, can be separated from the remaining monomeric subunits (and the 40-kDa subunit) by density gradient sedimentation. When dissociated with 0.36 M KI, 2.5 mM ATP, and 2.5 mM MgSO4, and added to membranes from which V1 has been dissociated, this V1(-40 kDa) subcomplex is able to reassemble with V0 to give a (H+)-ATPase with a proton pumping activity approximately half that obtained in the presence of the 40-kDa subunit. The undissociated subcomplex is not competent for assembly of a functional (H+)-ATPase. Interestingly, the monomeric fraction obtained from density gradient sedimentation contains the 40-kDa subunit but lacks the 34-kDa subunit. This monomeric fraction is nevertheless also able to assemble with V0 to give a functional proton pump. The V1V0 complexes assembled in the absence of either the 40- or 34-kDa subunits, while active, are not stable to detergent solubilization and immunoprecipitation, suggesting that both of these subunits play a role in stabilization of the (H+)-ATPase complex. Evidence for interaction between the 40- and 33-kDa subunits is also presented.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.     

H+/ATP stoichiometry of proton pump of turtle urinary bladder

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.     

An electrogenic proton pump associated with the Golgi apparatus of mouse liver driven by NADH and ATP

Golgi-apparatus membranes, isolated from mouse liver, pump protons inwards, when supplied with NADH or ATP. The acidification of Golgi-apparatus cisternae and vesicles was detected with neutral red, a permeant dye, as a difference in absorbance at 550 nm minus that at 600 nm. The maximum rates detected with NADH and ATP were between 0.0006-0.0009 and 0.0030-0.0050 delta OD units/mg of protein/min, respectively, at pH 7.5. The outside buffer used was a bovine serum albumin suspension. The acidification of Golgi apparatus was inhibited from 45 to 100% by ionophores and from 22 to 100% by uncouplers. The results implicate both ATP and a redox system coupled to NADH oxidation in the acidification of Golgi-apparatus membranes.     

Reversibility of coated vesicle dissociation

The dissociation of the coated vesicles to clathrin and uncoated vesicles and their reassociation have been studied under various conditions. The extent of reassociation is pH dependent and increases slightly with increasing concentrations of the components. Unlike the self-association of clathrin which is strongly salt dependent, the reassociation of clathrin and uncoated vesicles is practically independent of salt concentration. The coated vesicle gradually loses its coat with increasing pH, and the dissociation process is not an all or none reaction. Ca2+ inhibits dissociation of the coated vesicles and enhances the reassociation of clathrin and uncoated vesicles. Our results show that, although many conditions result in reassociation of protein and lipid vesicle, few conditions result in vesicles of both the same size and composition as native coated vesicles.     

Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.     

ATP4A gene regulatory network for fine-tuning of proton pump and ion channels

The ATP4A encodes α subunit of H⁺, K⁺-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H⁺, K⁺-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H⁺, K⁺ and Cl^?) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H⁺, K⁺-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H⁺, K⁺-ATPase on the basis of their affinity between drug–protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers.     

The association of clathrin fragments with coated vesicle membranes

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.     

Interaction of proton pump inhibitors with cytochromes P450: consequences for drug interactions.

Omeprazole, lansoprazole and pantoprazole are metabolized by several human cytochromes P450, most prominently by CYP2C19 and CYP3A4. Only pantoprazole is also metabolized by a sulfotransferase. Differences in the quantitative contribution of these enzymes and in the relative affinities of the substrates explain some of the observed interactions with carbamazepin, diazepam, phenytoin and theophylline and of the impact of the CYP2C19 (mephenytoin) genetic polymorphism. Of these drugs, pantoprazole has the lowest potential for interactions, both in vitro and in human volunteer studies.     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.     

Interaction of anions with iron-transferrin-chelate complexes.

Preliminary evidence suggested that phosphate or borote destabilize iron-ovotransferrin-nitrilotriacetate complexes in the absence of added bicarbonate. The iron-ovotransferrin-EDTA complex was prepared in the absence of bicarbonate, and a number of anions, including phosphate, sulfate, and citrate, were found to perturb the visible absorbance (lambdamax = 490 nm) of this complex. Other anions, such as chloride, nitrate, and perchlorate, had little or no effect on the spectrum. Also, when bicarbonate was added to a solution of the iron-transferrin-EDTA complex (A515 = 0.45), within 2 min, the visible absorbance had decreased to A515 = 0.13. Slowly a new peak appeared (lambdamax = 470 nm), evidently the iron-transferrin-CO3 complex. When these spectral changes were monitored in detail, the lack of an isosbestic point indicated the existence of one or more intermediates in the conversion of iron-transferrin-EDTA complex to the iron-transferrin-CO3 complex. Experiments using ternary complexes containing either 59Fe or [14C]EDTA show that both iron and EDTA nearly completely dissociate from the protein (most likely concomitantly within 2 min after bicarbonate is added. These observations are best explained by a paradigm which includes anion binding to the apoprotein. It is clear that there is an intimate relationship between anions and the binding of iron chelates by transferrin.