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SEVERAL TOPICS CONCERNING Na, K-ATPase

Makoto NAKAO,     Tokyo Medical and Dental University, Yushima, Bunkyo, Tokyo, Japan

Publisher Summary

This chapter discusses several topics concerning Na, K-ATPase. Na, K-ATPase is an entity that exchanges Na+ and K+ ions across the cell membrane against an electrochemical gradient, which is accompanied by ATP hydrolysis. This enzyme has many characteristics, some of which are specific for this enzyme. The enzyme binds ATP at a ratio of 1:1. The binding is not affected by the presence of Mg or Na, but K increases the dissociation constant and Na antagonizes the effect of K. The binding site is closely related to the active site according to Post, but there have been some experiments suggesting that ATP also acts aliosterically with Na on p-NPase activity. In the presence of a small amount of magnesium ion, or with NEM-treated enzyme, an ADP-ATP exchange react ion is observed and the reaction is accelerated by Na. The fact that cardiac glycosides specifically inhibit both cation active transport and Na, K-ATPase has been taken as strong evidence for the physiological entity of the enzyme in the active cation transport. The ratio between maximum binding of ouabain and the E-P in the presence of Na and Mg is 1:1.

Na,K-ATPase is an entity which exchanges Na+ and K+ ions across the cell membrane against an electrochemical gradient, accompanied by ATP hydrolysis. This enzyme has many characteristics, some of which are specific for this enzyme. Many good reviews (1–4) have been published recently on this subject. Therefore, this paper will deal very briefly with recent progress and problems concerning the study of this enzyme as well as with brief prospects of possible future developments.

Since Skou discovered Na,K-ATPase in crab nerve microsome fraction and proposed its role in the active transport of sodium and potassium ions, a number of papers on the study of this enzyme have been published, and its physiological significance has been extensively studied. At present, the purification of this enzyme, which is a cell membrane protein, is about to take place after a great many attempts by various investigators. A new stage in the investigation of this enzyme is about to begin.

Localization

Although microsome fractions are in fact used as an enzyme source, localization is restricted to cellular membranes of animal cells. Some papers (2) reported that certain bacteria have a slight Na,K-ATPase activity, but the enzyme activity has not yet been characterized precisely. Although it is very difficult to prove the non-existence of this enzyme in various cellular organells, such as nucleus, mitochondria, microsome, etc., almost all investigators believe that the enzyme is located in the cell membrane. Moreover, there is no Na,K-ATPase in the plant kingdom or in single cell animals except for some ouabain sensitive ATPase activity, which is, however, different from the Na,K-ATPase in ion-requirements (5–7). In higher animals in which nerves and muscles are differentiated, all cells show the activity. However, not all parts of the cell membrane of a cell show the activity. The part which faces outside the body does not show this activity but the part which faces inside the milieu interne shows the activity. This is best seen in the intestinal epithelium membrane. The microvilli side of the cell membrane in a cell does not contain the Na,K-ATPase, but the basolateral side of the membrane contains considerable activity (8). Periplasma in some bacteria may be regarded as a sort of milieu interne which may have developed phylogenetically during a long period of evolution, if this enzyme is actually present in bacteria.

Purification

Na, K-ATPase is a typical membrane protein which tranverses the membranes. It is extremely difficult to purify native membrane proteins from animal cells. After a great many trials in various laboratories considerable improvements in the purification method have been achieved (8–9). The purification factors are in the order of 100 microsome fractions from various animal organs. Although the assay conditions differ from laboratory to laboratory, maximum specific activities expressed as μ moles iP liberation/hour/mg protein, are as much as 7,000 (Nakao et al.) 1,500-2,000 (Hokin, Skou, Jørgensen, Schwartz) and 800 (Kyte). Materials used are ox brain, pig brain, dog kidney, electric organs from electric eels, etc. SDS-polyacrylamide electrophoresis shows one band [Nakao (18)] and two bands [Hokin (14), Schwartz (16), Kyte (19)]. However, it seems to be the consensus of most of the investigators in the field that P³² -protein, which is intimately related to one of the intermediates, shows a peptide with a molecular weight of about 100,000. The peptide is not divided into smaller peptides even after strong reduction or sonication in SDS. The amount of a peptide which accompanies the ³²P-peptide in most cases, is different in various laboratories and in various materials. The ratio of the amounts (the peptide/ a peptide of M.W.100,000) was 1:1 (Schwartz) 1:2 (Hokin) 1:1 (Kyte) and 0:1 (Nakao). Therefore, the necessity of the small peptide subunit for the activity has not yet been determined.

Substrate Specificity

This enzyme is relatively specific for ATP. dATP is attacked less strongly, followed by CTP(20). UTP, and ITP are hydrolyzed very slowly. p-Nitrophenylphosphate, (21–22) acetylphosphate, (22) carbamylphosphate (22) and umberipherone phosphate (23) are hydrolyzed in the presence of magnesium and potassium ions and the action is inhibited by ouabain. There is no doubt about the identity of the enzyme proteins (or at any rate the main component) of the Na,K-ATPase and K-p-Npase but the precise behaviour of ouabain inhibition, such as the interaction of Na and K ions with the enzyme, the amount of phosphorylated intermediates and the competition of both substrates are complex. A P³² -enzyme protein is obtained by incubation of the enzyme at 0° or 37°C with γ-ATP³² in the presence of Mg and Na ions (24). The amount of the P³²-enzyme (E-P) decreases with further addition of potassium ion. The E-P³² shows an acylphosphate nature (25–26) when the reaction is stopped with acid or SDS at neutral pH. The P³² is undoubtedly bound to a peptide with a molecular weight of about 100,000. The experiment of Post (27) indicating that the binding side of P³² is an aspartic residue is in opposition to the claim of Hokin’s(28–29), that the site consists of a glutamic residue. Some evidence has already been accumulated to support the theory that the E-P is an intermediate of the enzyme reaction (30), but the protein obtained by the addition of acid or SDS is a denatured enzyme (31) different from the true intermediate. This fact might cause some confusion. When p-nitrophenol is used as substrate, a sufficient amount of E-P can not be detected.

Binding of ATP and ADP-ATP Exchange

The enzyme binds ATP at a ratio of 1:1 (32,20). The binding is not affected by the presence of Mg or Na, but K increases the dissociation constant and Na antagonizes the effect of K. The binding site is closely related to the active site according to Post, but there have been some experiments suggesting that ATP also acts allosterically with Na on p-NPase activity (34). In the presence of a small amount of magnesium ion, or with NEM-treated enzyme(35), an ADP-ATP exchange reaction(35,36) is observed and the reaction is accelerated by Na.

Ouabain Binding

The fact that cardiac glycosides specificially inhibit both cation active transport and Na,K-ATPase has been taken as strong evidence for the physiological entity of the enzyme in the active cation transport. A Ki value between 10−6 and 10−8 M is obtained in most cases, but the apparent Ki values vary widely from one animal species to another and to a lesser extent from one organ to another. The ratio between maximum binding of ouabain and the E-P in the presence of Na and Mg is 1:1. The rate of binding varies according to the conditions Matsui and Schwartz(39). Mg+++Na++ATP, and iP+Mg++ show the greatest binding followed by Na++ATP, and Mg++. In the presence of inorganic P³² or the phosphate and magnesium ion, ouabain is bound to the enzyme, and at the same time the inorganic phosphate is bound to the enzyme. The resulting phosphorylated protein which is obtained by the addition of acid can not be distinguished from the E-P which comes from ATP³²(38). This finding suggests a conformation change induced by the non-covalent binding of ouabain. This suggestion is consistent with the finding of Schwartz’s(1) laboratory, that a change was detected using hydrophobic probes, C D spectrography, and a ³H exchange reaction. The latter findings were obtained using a partially purified enzyme, of which the content was supposed to be only 5-10% on the basis of protein. They assumed that great changes in conformation occur. Further experiments are expected.

Relation to lipid

It is considered that the enzyme contains a great amount of phospholipids and when solubilized with a detergent, the phospholipids in the solubilized particles are replaced by a great amount of the detergent (38). It might be, therefore, pointless to try to obtain the precise molecular weight. There is still some confusion regarding the specificity of phospholipids. Acidic phospholipids are reported to play an important role in the restoration of the enzyme activity, but quantitative intensity and specificity differ from a report to another (39–42). These variations seem to depend on a procedure in which the phospholipid component was eliminated and on a preparation used which is still impure. At any rate, the dephosphorylation step was strongly affected by the treatment compared with the phosphorylation step (43).

Reaction mechanism

A great number of papers have been published on the kinetic analysis. Most of them are omitted from this minireview due to the limitations of space. Some years ago, Glynn and his associates succeeded in the phosphorylation of ADP using very steep concentration gradients of Na+ and K+ across red cell membranes (and E-P, which differ from the former in the order of ADP-release etc. This theory was proposed as a part of Tonomura’s general scheme concerning energy transformation. Japanese and European investigators, including Skou and Repke, seem to prefer Tonomura’s theory. Repke proposed a Flip-flop model (4,47,48) which consists of two identical subunits, whether or not each subunit consists of one peptide or two peptides. According to this theory the reaction in one subunit is tightly coupled with the identical reaction in the other subunit, which follows the reaction by a delayed half phase. This beautiful hypothesis can explain many confused phenomena including allosteric effects of Mg, ATP, Na and K and physicochemical data. The principle of this theory may influence models of the molecular movements not only in the field of active transport but also in the other fields of energy transformation.

Posture of the Enzyme in Intact Membranes

The active site of the enzyme exists within the cell and the ouabain site is assumed to be located on the outer surface of the cell, but this theory lacks decisive evidence. Na+ and Mg++ activate the enzyme inside the cell and K+, outside (49). A Hg compound in which PCMB is bound covalently to a dextran with a molecular weight of 250,000 (50) when applied to intace red cells, inhibits the active transport, and the Na,K-ATPase activity when measured after washing and isolation of the membrane. Therefore, the enzyme faces outside the cell. According to Baker(51), the inhibition of the active transport in intact Hela cells by ouabain was strictly competitive with the concentration of K+ in the medium. This finding is quite different from the data using isolated membranes or partially purified enzyme preparation(1). It may enable modification of the enzyme specifically from the stereochemical point of view.

Biological Significance

1) The main role of the Na,K-ATPase is, of course, the active exchange transport. Three sodium ions from inside to outside and two potassium ions from outside to inside move with hydrolysis of one ATP (52). The view that this active transport results in completing the preparation for excitation and active transport of some amino acids, sugars, etc. is generally accepted (2,3).

2) The decrease in ATP and the increase in iP and ADP affect the energy metabolism and accordingly the rate of glycolysis or respiration (53, 54).

3) The movement of cations is electrogenic, but in a steady-state, protons are reabsorbed to synthesize ATP by glycolysis or respiration. Therefore OH- or Cl− (or proton) moves to maintain electroneutrality at both sides of the cell membrane. The species of ion which compensates for the charge difference may vary from one organ to another and from one species to another. The movement of neutral salts accompanys the movement of water, but the amount may not be appreciable.

4) The activity is controlled by various factors. The first factor is the concentrations of the substrate (ATP) together with the products (ADP and inorganic phosphate) and the accelerators (Mg, Na and K). In this case, position (whether outside or inside the cell) is crucial. Physiological substances, such as vitamins [A,C (55) and E(56)] free fatty acids, and hormones inhibit Na,K-ATPase activity, but almost all of these experiments were conducted, using crude preparations. Therefore, it is possible that the effect is mediated indirectly by the third factor or factors. Application of antibody and reconstitution of the membrane with partially purified enzyme have attracted the attention of many investigators (4). However, characterization of purified enzyme may be the more urgent problem at the present stage of investigation.

References

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FUNCTION AND ORGANIZATION OF CHROMAFFIN VESICLE

Norman Kirshner,     Department of Biochemistry, Duke University Medical Center, Durham, N. C. 27710

(Received in final form 26 December 1973)

Publisher Summary

This chapter describes the function and organization of chromaffin vesicle. The vesicles contain the highest concentration of adenine nucleotides, about 0.12 M, known to occur in any tissue or organelle. ATP is the major nucleotide component accompanied by smaller amounts of ADP and AMP, but the relative amounts vary in different species. The presence of small amounts of GTP, UTP, and mucopolysaccharides has also been reported. The concentration of CA within the vesicles is estimated to be 0.55 M. The molar ratio of CA: adenosine phosphates are close to 4:1 providing electrical equivalence at physiological pH within the vesicle. Disk-gel chromatography of the soluble proteins shows seven or eight major components and a similar number of minor components. Dopamine-3-hydroxylase is present both as a constituent of the membrane and as a constituent of the soluble interior milieu of the vesicles. In bovine and rabbit vesicles, 40 to 45% of the enzyme activity remains with the membrane after several washes, while in rat 80 to 85% of the activity remains with the vesicles.

Chromaffin vesicles of the adrenal medulla were first isolated more than twenty years ago and shown to contain most of the adrenaline and noradrenaline present in the gland (1,2). Subsequent studies have indicated that very little of the catecholamines exist in the cytoplasm (3). During the past twenty years it has become apparent that chromaffin vesicles are not merely passive storage organelles but are structures highly organized to take up, synthesize, store, and secrete catecholamines (CA). It is the purpose of this discussion to examine each of these functions in relation to the current status of our knowledge of the composition and organization of the chromaffin vesicle.

Composition of Chromaffin Vesicles

Highly purified chromaffin vesicles of the adrenal medulla can be isolated by a combination of differential and density gradient centrifugations. The overall composition of the vesicle is shown in Table 1. The vesicles are readily lysed upon suspension in distilled water or dilute buffer and after several washes practically all of the CA, adenosine phosphates, other small molecules and 75 to 80% of the total protein are solubilized. It is convenient to consider the soluble and particulate components separately.

TABLE 1

Composition of Bovine Adrenal Storage Vesicles

A Soluble Components

The vesicles contain the highest concentration of adenine nucleotides, about 0.12 M, known to occur in any tissue or organelle. ATP is the major nucleotide component accompanied by smaller amounts of ADP and AMP but the relative amounts vary in different species. The presence of small amounts of GTP, UTP (4) and mucopolysaccharides (5,6) have also been reported. The concentration of CA within the vesicles is estimated to be 0.55 M. The molar ratio of CA:adenosine phosphates is close to 4:1 providing electrical equivalence at physiological pH within the vesicle.

Disc-gel chromatography of the soluble proteins shows 7 or 8 major components and a similar number of minor components. Two of the proteins have been characterized – dopamine-β-hydroxylase [EC 1.14.2.1] (DBH) which accounts for 5 to 7% of the protein and chromogranin A which represents 30 to 35% of the protein. The water soluble proteins have been named chromogranins but DBH should be excluded from this classification since it appears to be entirely different from the other proteins. Although a considerable amount of work has been done on chromogranin A there are some puzzling aspects of its structure and behavior in solution which require additional studies to understand (7). The remaining chromogranins have been characterized only to the extent that they are acidic proteins having an isoelectric point around pH 4.5, and that the amino acid composition of the mixture of chromogranins is very similar to the amino acid composition of chromogranin A.

B Membrane Components

The membrane of the chromaffin vesicle contains approximately 20% of the total vesicle protein and practically all of the lipid. On a dry weight basis the membrane of bovine adrenal vesicles contain 36% protein and 64% lipid. Approximately 23% of the total lipid is cholesterol and the remainder is a mixture of phospholipids containing an unusually high (15%) content of lysolecithin. Proteins which have been identified as constituents of the vesicle membrane are DBH, ATPase, cytochrome b 561 and a cytochrome b:NADH reductase. Gel electrophoresis of the membrane proteins after solubilization in sodium dodecyl sulfate (SDS) and mercaptoethanol shows the presence of 15 peptides with a pattern quite distinct from that of mitochondria and microsomes (7).

Winkler and co-workers (8) have preposed the generic name chromomembrins for the protein constituents of the vesicle membranes. In their studies in which the membranes were solubilized in either phenol-acetic acid-urea or in SDS without the addition of mercaptoethanol they identified two major peptide components – chromomembrin A and chromomembrin B. Chromomembrin A appears to be identical with DBH while immunological studies with chromomembrin B suggest that this protein is localized on the exterior of the vesicle membrane (8).

Chromogranin A has been reported to be a major constituent of the vesicle membrane (9) but other studies (7,8) indicate that it represents less than 10% of the total membrane protein and may be present as a contaminant.

Dopamine-β-hydroxylase is present both as a constituent of the membrane and as a constituent of the soluble interior milieu of the vesicles. In bovine and rabbit vesicles 40 to 45% of the enzyme activity remains with the membrane after several washes while in the rat 80 to 85% of the activity remains with the vesicles. This of course raises questions. Are the soluble and membrane bound enzymes different in structure? Is the soluble form of the enzyme derived from the membrane enzyme or vice versa? What structural properties of the enzyme enable it to become firmly associated with the membrane? What is the physiological significance of the distribution? Gel electrophoresis studies of the membrane bound and soluble form of the enzyme indicate no differences between the two. The amino acid composition of the soluble and membrane bound enzymes have been reported to be similar by two laboratories (10,11). The data agree well within each of the laboratories but there are several large discrepancies in the reported amino acid compositions between the groups. In addition a third study (12) of the amino acid composition of DBH shows ever more significant differences but it is not clear whether the purified DBH used in the latter study was the soluble form only or a mixture of the soluble and membrane bound enzymes. The data indicate that significant contamination occurred in one or more of the preparations and further work is necessary to determine whether the amino acid composition of the enzymes are identical.

The subunit structure of DBH has been reported from several laboratories (8,12,13). The native enzyme has a molecular weight of 290,000 determined by sedimentation equilibrium in 0.1 M sodium chloride. In 6 M quanidine HCl the molecular weight is 150,000 to 160,000 and in 6 M quanidine HCl containing mercaptoethanol or dithiothreitol the molecular weight is 77,000. The subunit molecular weight of 77,000 has also been confirmed for both the soluble and membrane bound enzyme by gel electrophoresis after treatment with 1% SDS – 0.1% mercaptoethanol. The enzyme thus consists of subunits joined in pairs by disulfide bonds and two such units held together by non-covalent forces. Recently Wallace et al. (13) have shown DBH to be a glycoprotein containing about 4% carbohydrate which consists of residues of mannose, glucosamine, galactose, fucose and sialic acid.

Role of ATP and Chromogranins in the Storage of Catecholamines

Studies of isolated chromaffin vesicles led to the concept that CA were maintained within the vesicle in a non-diffusible complex consisting of catecholamines, ATP and chromogranins (14). Physical chemical studies utilizing NMR spectroscopy and titrimetric methods indicated that only weak interactions between CA and ATP occurred in solutions and binding of CA to the chromogranins also could not account for the amount or stability of the amines within the vesicles. However other types of physical chemical studies have yielded more positive results. High speed centrifugation of mixtures of catecholamines and ATP results in the formation of sedimentable high molecular weight complexes of CA and ATP. Addition of low concentration of bivalent cations increases the degree of aggregation and upon dilution of solutions of CA and ATP (17% w/v CA and molar ratio of CA:ATP of 3:5) phase separation occurs in the presence of Ca++. Chromogranins and gelatin sediment with the multimolecular complexes of CA and ATP but serum albumin does not. Osmolality and fluorescence measurements confirm the existence of intermolecular bonds, and fluorescence quenching and shortening of the fluorescence halftime shows that mobility within the complex is relatively high indicating a non-rigid reversible binding (14).

The picture that emerges from these studies is that the CA, ATP and chromogranins interact through weak intermolecular forces which are rapidly made and broken forming a complex which arises not from the stability of the interaction per se but rather from the statistical kinetics of the interactions. That is, one has a highly mobile complex in which CA, ATP and chromogranins are continuously interchanging partners and these interactions lead to a greatly reduced osmolarity within vesicle. In reconstituted systems the degree of aggregation measured by sedimentation velocity increases with decreasing temperature. At lower temperatures more stable interactions may occur and this may explain the observations that even though catecholamines can cross through the membrane at 0° they are not incorporated into the storage complex.

Role of Membrane Proteins in the Uptake of Catecholamines – Evidence for Amine Transport Utilizing Membrane ATPase and an amine carrier

More than ten years ago it was demonstrated that isolated chromaffin vesicles can incorporate exogenous amines into their storage pools at 37° but not at 0°. This uptake is temperature-sensitive: at 37° the incorporation is twofold that at 30°, it is stimulated several fold by Mg++-ATP, and it is inhibited by reserpine and a variety of other drugs, by sulfhydryl agents such as N-ethylmaleimide, and by chelating agents such as EDTA. The vesicle membrane has a Mg++-ATPase activity which is also inhibited by EDTA and by NEM but not by reserpine. The EDTA inhibition of both the ATPase activity and incorporation can be overcome by excess Mg++. The membranes also contain a Ca++-ATPase but this activity is much less sensitive to inhibition by NEM, and Ca++ does not stimulate incorporation of CA. These data indicate that an enzymatic mechanism involving the ATPase as well as another event which is inhibited by reserpine is involved in the uptake. In these studies the accumulation of a radiolabeled CA was measured and one could not determine whether transport across the membrane, incorporation into the storage pool or both were sensitive to reserpine and NEM.

Another method of observing the uptake phenomenon is to determine the rate of release of endogenous amines in the presence or absence of Mg++-ATP and in the presence or absence of inhibitors of uptake. These types of studies have shown that the efflux rate in the absence of ATP was much faster than the influx but in the presence of ATP the rate of influx was increased to equal the rate of efflux. In the presence of ATP and NEM the efflux and influx rates were the same as that in the absence of ATP. Similar results have been obtained using reserpine as an inhibitor. In the presence of Mg++-ATP the t 1/2 for depletion was four times as long as that in the absence of Mg++-ATP but in the presence of both Mg++-ATP and reserpine the t 1/2 was the same as that in the absence of Mg++-ATP. Thus reserpine did not affect either the stability of the storage pool or the rate of efflux but did inhibit transport of the amines into the vesicles.

A third method for studying transport across the membrane utilizes vesicles reformed from membranes of hypotonically lysed chromaffin vesicles. The reformed vesicles can accumulate catecholamines from the surrounding medium in the presence of Mg++-ATP against a concentration gradient as high as 140:1. As with intact vesicles the uptake is inhibited by NEM and by reserpine (14,15).

Intact vesicles used for uptake studies are contaminated to various degrees with ATPases from mitochondria and microsomes but these appear to be much less sensitive to NEM than does the vesicle ATPase. This is evident from the fact that concentration of NEM which completely inhibit uptake in intact vesicles inhibit the ATPases by only 25-50%. However if one uses membranes from vesicles purified by density gradient centrifugation NEM inhibits the ATPase activity 80-90%. In intact vesicles ATP hydrolysis correlates well with uptake when both are measured as the differences in activities in the presence and absence of NEM. Under these conditions 1.2 to 1.5 moles of ATP are split per mole of catecholamine taken up (16). With reformed vesicles the rate of ATP hydrolysis is 200 times faster than the rate of influx but since these vesicles do not have a stable storage complex much of the amine transported into the vesicle probably leaks out during the experimental period.

The evidence cited above indicates an ATP-utilizing transport system for catecholamines, and, as in other translocation systems, suggests a membrane carrier may be involved. Studies of binding of various aromatic amines to membranes of purified chromaffin vesicles show the presence of a high affinity binding site which exhibits the same rank order of specificity-serotonin > adrenaline > metaraminol – as do intact vesicles for uptake. The binding of the amines was affected by ATP, Mg++ and reserpine in a manner which cannot be readily interpreted at the present time; neither Mg++ nor reserpine by themselves inhibited binding but together they caused a 30% inhibition; ATP by itself caused a 35% inhibition which was not affected by Mg++ and the inhibitions caused by ATP, reserpine and Mg++ were additive. These data are suggestive and additional work is clearly required to establish the existence of a membrane-bound amine carrier. Recently Slotkin (17) has proposed a model for the uptake of catecholamines into adrenal chromaffin vesicles which provides a basis for further experiments.

Studies of the uptake of various amines revealed the presence of a second uptake system (18,19). This system is insensitive to stimulation by Mg++-ATP and insensitive to inhibition by reserpine. The rank order of specificity for this system appears to be in reverse order – metaraminol > tyramine > adrenaline to the ATP-mediated uptake. It is only poorly defined at present and its significance is obscure.

Biosynthesis of Catecholamines

The prevailing dogma on the biosynthesis of the catecholamines states that dopa and dopamine are enzymatically derived from tyrosine by the sequential actions of tyrosine hydroxylase and aromatic-L- amino acid decarboxylase localized externally to the chromaffin vesicle. Dopamine is transported into the vesicle where it is converted to noradrenaline by DBH. The noradrenaline is incorporated into the storage pool and subsequently released into the cytoplasm (some of the noradrenaline may be released without entering the storage pool) where it is methylated to form adrenaline by phenylethanolamine-N-methyltransferase (PNMT). The adrenaline is then taken up by the vesicles and incorporated into the storage pools. This scheme is based on the observations that the enzymatic sites of both the soluble and membrane bound dopamine-β-hydroxylase are exposed only to the interior of the vesicle (20) and that PNMT which is localized in the cytoplasm has a high order of specificity for β-hydroxylated phenylethylamines (21). An alternative to this pathway has been proposed by Laduron (22) who suggests that the specificity of PNMT is not as rigid as initially proposed and that dopamine is methylated to epinine in the cytoplasm prior to its entry into the chromaffin vesicle where it can be then directly oxidized to adrenaline. It has been further suggested from immunologic studies that some of the membrane bound DBH is exposed to the cytoplasm (23). If this is so then noradrenaline may be formed on the cytoplasmic side of the chromaffin vesicles where it may be methylated to adrenaline prior to uptake. These proposals are interesting but require considerably more experimental evidence to establish their validity and