Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 ± 0.011 mM and 129.3 ± 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 ± 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 ± 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture. Mol. Reprod. Dev. 50:443–450, 1998. © 1998 Wiley-Liss, Inc.     

Properties of the calcium-sensitive components of bovine arterial actomyosin.

A calcium-sensitive actomyosin was prepared from bovine aortic muscularis. Calcium binding to this arterial actomyosin was measured using a centrifugation method. The amount of bound calcium necessary for full activation of the arterial actomyosin adenosine triphosphatase was approximately 36 μmol of Ca²⁺/kg of aorta. Calcium stimulation of the actomyosin ATPase could be prevented by lowering the free magnesium from 7 to 1 mm. However, calcium binding to the actomyosin increased slightly with a reduction of free magnesium levels. Positive cooperativity was evident in the sequence of reactions beginning with the binding of calcium and ending with the hydrolysis of ATP. However, there was no evidence for the cooperativity occurring at the initial calcium-binding step.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr ? 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.     

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

Ionic and substrate requirements of the high affinity calcium pumping ATPase in endoplasmic reticulum of pancreas

1. Calcium transport and ATPase activities were determined in microsomal vesicles from pancreatic tissue enriched in endoplasmic reticulum membranes. 2. Calcium transport and ATPase share the following properties: (i) magnesium was required with a K0.5 of 0.7 mM and maximal pumping ATPase activity at 5 mM Mg-ATP; (ii) at saturating magnesium concentrations, calcium increased ATP splitting activity up to three times with an apparent K0.5 close to 0.3 microM calcium; (iii) potassium stimulated the high calcium affinity Mg2+-dependent ATPase and calcium transport. 3.The properties of the calcium pumping system fulfil the cationic and substrate requirements from a physiological point of view.     

Structural and physiological effects of calcium and magnesium in Emiliania huxleyi (Lohmann) Hay and Mohler

In organisms which perform both photosynthesis and calcification, the fact that calcification proceeds faster in the light than in the dark has led to the long-established view that photosynthesis and calcification are closely coupled. It is now clear that calcification does not promote photosynthesis, but an enhancement of calcification by photosynthesis could still explain why calcification is faster in the light. To test this, the kinetics of the two processes were monitored over a wide range of calcium concentrations (0-50 mM) in the coccolithophore Emiliania huxleyi. The addition of 50 mM calcium strongly inhibited both processes, but when incubated in lower concentrations, rates of calcification increased up to 20 mM calcium whilst those of photosynthesis remained constant over the same range of calcium concentrations. So, rates of calcification are able to rise without a concomitant increase in photosynthetic rates. In addition, calcification rate and coccolith morphology responded similarly to changes in calcium concentrations; low calcification rates were associated with poor coccolith structure (undercalcification) and high calcification rates with perfectly formed coccoliths. Calcium concentration thus strongly influences calcification affecting both crystal structure and rate of calcite deposition. A similar structural analysis of coccoliths from cells grown in different magnesium concentrations showed that this ion is also essential for calcification, since strong signs of coccolith malformation and undercalcification were apparent at both low and high magnesium concentrations. In contrast with the calcium results, coccoliths were flawless only in the normal seawater concentration of 58 mM magnesium. We conclude that photosynthesis and calcification are not closely coupled and that calcification depends on a precise balance of both calcium and magnesium concentrations.     

Formation of magnesium-phosphoenzyme and magnesium-calcium-phosphoenzyme in the phosphorylation of adenosine triphosphatase by orthophosphate in sarcoplasmic reticulum. Models of a reaction sequence

The aim of the present study was to test simple reaction sequences which describe calcium-independent plus calcium-dependent phosphorylation of sarcoplasmic reticulum transport. ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg . E . Pi), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium-phosphoenzyme (Mg . E-P). Phosphorylation, performed at pH 7.0 20 degrees C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent magnesium half-saturation constant and an increase in maximum phosphoprotein formation with increasing calcium loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent orthophosphate half-saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X-537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low-affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation. Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E . Mg . Pi or Mg . E-P or to both, but neither to E. Mg nor to E . Pi. Secondly, calcium-independent and calcium-dependent phosphoproteins are magnesium-phosphoenzymes. Calcium-dependent phosphoenzyme is a magnesium-calcium-enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg . E-P . (Cai)2], and not a 'calcium-phosphoprotein' without bound magnesium.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

Ionic and nucleotide requirements for microtubule polymerization in vitro.

The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.     

Effect of cardiac and skeletal tropomyosin on Mg2+-actomyosin ATPase.

Tropomyosin, one of the proteins regulating the sarcomere, was prepared from pig heart and rabbit skeletal muscles. The effect of these two different tropomyosins was studied between 0.5 and 10 mM of Mg2+ at a constant ATP concentration (1 mM) on reconstituted actomyosin prepared from pig heart myosin and rabbit skeletal actin. Cardiac and skeletal tropomyosin both activated the ATPase at low Mg2+ concentrations and inhibited it above 3 mM. The pig heart and rabbit skeletal tropomyosins which contain two isomers, alpha alpha and alpha beta, respectively has very similar effects on actomyosin ATPase.     

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM · (Ca²⁺)₄ is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01–1 μ M) was 0.9 nM; the respective apparent dissoclation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1–50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by ¹²⁵I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM · (Ca²⁺)₄. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E · CaM) is at least 100-fold greater than the apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex, as judged from non-saturation ¹²⁵I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

The phosphonic acid analog of phosphatidylglycerol phosphate: influence on Escherichia coli growth and physiology.

At 20 microM, rac-3,4-dihydroxybutyl-1-phosphonate (DBP) has only a slight bacteriostatic effect on Escherichia coli. However, cells lose viability when the medium also contains either 20 mM magnesium or calcium ions. Magnesium ions stimulate the incorporation of DBP into (1,2-diacyl)-sn-glycerol-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-pho sphonate, the phosphonate analog of phosphatidylglycerol phosphate. Much higher DBP concentrations are needed to block the growth of a pgsA3 mutant than to block the growth of an isogenic wild-type strain. The DBP-treated pgsA mutant also has a much higher survival rate when stored in the cold than does the DBP-treated wild-type strain. Furthermore, the pgsA3 mutant grows normally in the presence of DBP and magnesium ions. Treatment with DBP and magnesium ions does not appear to disrupt the cell's inner or outer membranes. However, it does block macromolecular and phosphoglyceride synthesis. A combination of 20 microM rac-DBP and 0.5 mM spermidine or 0.125 mM spermine is bacteriostatic. These studies indicate that the PGP analog contributes to DBP's bacteriostatic effect when the growth medium contains low concentrations of magnesium or calcium ions and is responsible for its bactericidal effect when the medium contains high concentrations of these ions.     

Regulatory proteins of lobster striated muscle.

The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.     

Actin mediated calcium dependency of actomyosin in a myxomycete

A fraction obtained from $\text{Physarum polycephalum}$ by differential centrifugation displays magnesium adenosine triphosphatase activity; at low ionic strength (0.07 M KCl) the rate at which ATP¹ is split in 0.1 mM CaCl₂ is from 1.5 to 6.6 times the rate in 1 mM EGTA¹. Both actin and myosin are present in this fraction. On SDS gels several polypeptide bands are present in the range of 39,000 daltons to 14,000 daltons as well as those of actin and myosin. The addition of desensitized rabbit muscle actin to the fraction increased the rate of ATP splitting in EGTA, thereby decreasing the EGTA inhibition 30–50%. We conclude that actomyosin regulation by calcium in this acellular slime mould is, at least in large part, mediated through actin.     

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.     

Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP

In the absence of added calcium, inhibition of NAD-specific isocitrate dehydrogenase by ATP occurred without ADP (I0.5 = 1.8 mM) and with 0.2 mM ADP3- (I0.5 = 1.0 mM) at subsaturating substrate concentrations at pH 7.4. Inhibition by ATP was competitive with NAD+ in the presence and absence of ADP and was not reversed by magnesium citrate. No reversal of ATP inhibition by free Ca2+ was observed in the presence of ADP (0.2 mM). However, when ADP was absent, increasing Ca2+ first caused progressive reversal of ATP inhibition followed by activation by ATP. Without ADP, the S0.5 for calcium activation was 80-140 microM at ATP concentrations between 0.6 and 3.0 mM. The S0.5 for ATP activation, in the absence of ADP, was 1.1 and 2.1 microM when free Ca2+ was held constant at 0.1 and 1.0 mM, respectively. As in activation by ADP, ATP decreased the S0.5 for magnesium isocitrate without affecting V. However, in contrast to ADP, the activation by ATP occurred without lowering the Hill coefficient for the substrate. GDP activated the enzyme at relatively high concentrations of Ca2+ but not without added Ca2+.     

Calcium sensitivity of vertebrate skeletal muscle myosin

The calcium sensitivity of vertebrate skeletal muscle myosin has been investigated. Adenosinetriphosphatase (ATPase) activity was assayed in a reconstituted system composed of either purified rabbit myosin plus actin or myosin plus actin, tropomyosin, and troponin. The calcium sensitivity of actomyosin Mg-ATPase activity was found to be directly affected by the ionic strength of the assay medium. Actomyosin assayed at approximately physiological ionic strength (120 mM KCl) demonstrated calcium sensitivity which varied between 6 and 52%, depending on the myosin preparation and the age of the myosin. Mg-ATPase activity was increased when calcium was present in the assay medium at physiological ionic strength. Conversely, actomyosin Mg-ATPase activity assayed at a lower ionic strength (15 mM KCl) was inhibited by addition of calcium. Addition of tropomyosin and troponin to the assay increased the calcium sensitivity of the system at the physiological ionic strength still further (up to 99% calcium sensitivity) and conferred calcium sensitivity on the system at the lower ionic strength (greater than 90% calcium sensitivity). A correlation also existed between myosin's calcium sensitivity and the phosphorylated state of light chain 2.     

Activation of snail (Helix pomatia) nervous tissue tyrosine monooxygenase by calcium in vitro.

Addition of calcium chloride to soluble preparations of tyrosine monooxygenase from snail brain appears to produce an activation of the enzyme when assayed with subsaturating concentrations of the pteridine cofactor 6 MPH4 (2-amino-4-hydroxy-6-methyltetrahydropteridine). While some increase in the activity occurs with calcium chloride at a concentration of 0.01 mM, activation is increased by about 100% at 1mM and reaches a maximum at 5mM (144%) where it remains more or less constant up to 10mM. Barium chloride also produces an activating effect although it is much less pronounced while magnesium chloride is without effect. EGTA has no direct effect on the enzyme but antagonises the activation produced by calcium chloride. The activation of tyrosine monooxygenase by calcium is reflected in changes in the kinetic properties of the enzyme, decreasing the Km from 43 muM to 19 muM for tyrosine and from 670muM to 230muM for the pteridine cofactor. No change was observed with V values for either tyrosine or pteridine cofactor. It is suggested that calcium, which enters the nerve terminal during nerve stimulation, regulates the transmitter dopamine by activating the rate-limiting enzyme tyrosine monooxygenase.