Eric A. Stein

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlated with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+ calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be inferred from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin X Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.

Pig kidney particulate aminopeptidase a zinc metalloenzyme

Abstract 1. 1. Particulate aminopeptidase (EC 3.4.1.2) from pig kidney, which contains 2 gatoms of zinc per mole protein as the only metal component (Wacker et al. (1971) Helv. Chim. Acta 54, s473–485) is converted to an inactive metal-free apoaminopeptidase by electrodialysis, gel filtration, or treatment with chelating agents. 2. 2. Metal depletion does not significantly affect the molecular weight and electrophoretic mobility of the protein, but reduces the resistance of the enzyme towards denaturation under extreme conditions. 3. 3. Zn2+, Cu2+, Co2+ or Ni2+, but no other cations, restore activity to the apoaminopeptidase. 4. 4. Restoration of enzyme activity is directly proportional to the amount of divalent metal ions added up to 2 gatoms per mole of apoaminopeptidase; there is no increase of activity upon further addition. 5. 5. The data indicate that particulate aminopeptidase from pig kidney is a zinc metalloenzyme, and suggest that both atoms are essential for activity

Calcium, magnesium and the conformation of parvalbumin during muscular activity

The conformation of perch parvalbumin in the Ca-, Mg- and metal-free state was studied by intrinsic fluorescence, trypsin susceptibility, thiol titration and circular dichroism. The data reveal that Ca-parvalbumin has a more compact structure than the metal-free protein, with a high alpha-helical content and a buried thiol. No difference in conformation could be detected between Mg- and Ca-parvalvumin, indicating that the Ca-Mg exchange that may take place during muscular activity is accompanied by little or no structural changes. Furthermore, recently published kinetic parameters can now be interpreted as meaning that, during the contraction-relaxation cycle, parvalbumin often stays in the Mg-form instead of switching to the Ca-form which is predominant in vitro.

A new solid-phase chelator with high affinity and selectivity for calcium: Parvalbumin-polyacrylamide

A solid-phase chelator for calcium was prepared by linking parvalbumin (a muscle calcium-binding protein of remarkable stability) to the polyacrylamide matrix of Bio-Gel P-60. The immobilized parvalbumin can be used repeatedly, and, due to its remarkable affinity for calcium, it is capable of lowering calcium concentration to ≤10−10m at neutrality. The affinity for calcium remains relatively high even at pHs as low as 4–5, at which complexants such as EDTA, EGTA, or Chelex would be quite inefficient. As immobilized parvalbumin binds Mg2+ with an apparent Kdiss 3.5 orders of magnitude higher than that of Ca2+, it can be used to control calcium concentrations even in the presence of magnesium. The affinity for calcium of any strong complexant can be determined by measuring Ca2+ distribution between this complexant and the solid-phase parvalbumin. Due to its outstanding affinity and selectivity for calcium, immobilized parvalbumin could prove to be a powerful tool in investigating the role of calcium in the regulation of metabolic processes.

Regulation of a presynaptic adenylate cyclase from bovine cerebellum by β-adrenergic receptors

Abstract Intact crude synaptosomes from bovine cerebellum contain, in addition to an externally accessible (postsynaptic) adenylate cyclase, an enzyme with its catalytic center oriented towards the inside of the synaptosome (presynaptic adenylate cyclase). This is demonstrated by the unmasking of latent adenylate cyclase activity by Triton X-100. Furthermore, intact crude synaptosomes can synthesize cyclic AMP from adenine. This synthesis takes place inside the synaptosome as the postsynaptic adenylate cyclase is inactive in the Krebs-Ringer buffer. Presynaptic adenylate cyclase activity is not influenced by depolarization, as shown by [ 3 H]adenine pulse-labeling, but is stimulated by (−)-norepinephrine and (−)-isoproterenol. (±)-Propranolol inhibits this stimulation whereas phentolamine has no effect, suggesting the presence of a β-adrenergic receptor-coupled presynaptic adenylate cyclase.

Sarcoplasmic calcium-binding proteins in protochordate and cyclostome muscle

SummaryA sarcoplasmic calcium-binding protein (SCP) has been purified from the muscle of the protochordate Amphioxus and shown to be more similar to invertebrate SCPs than to their counterpart found in vertebrates, i.e. parvalbumins. The Amphioxus protein has a pI of 4.9, is rich in tyrosine and tryptophan, has a molecular weight of 22,000 and binds strongly 2Ca2+ with a pK of 7.88. Magnesium competes with calcium for only one of the two metal-binding sites and induces positive cooperativity in Ca2+ binding.In cyclostome muscle (lamprey and hagfish), no protein with high affinity for Ca2+ or Mg2+ could be found, irrespective of molecular weight. Instead, a protein with moderate affinity for Ca2+ (⩽105m−1) was detected: it has a molecular weight of 60,000 and might be quite ubiquitous, as the presence of a similar protein has been reported both in red and white muscle of vertebrates such as chicken and rabbit.

Synergistic activation of bovine cerebellum adenylate cyclase by calmodulin and β-adrenergic agonists

The relationship between calmodulin-dependent and ?-adrenergic-sensitive adenylate cyclase activities was examined in membrane preparations from bovine cerebellum. Although stimulation by ?-adrenergic agonists or calmodulin can occur independently, it is shown that their simultaneous presence has a strong synergistic effect on enzyme activity. Calmodulin did not influence the regulatory components of the neurotransmitter-dependent pathway as shown by the lack of effect on (1) receptor affinity, (2) GTP requirement for receptor-mediated activation, (3) rate of activation by guanyl 5?-yl imidodiphosphate [Gpp(NH)p]. Conversely, isoproterenol and guanine nucleotides did not modify to a significant extent the characteristics of enzyme stimulation by Ca(2+) and calmodulin. Furthermore, calmodulin and Gpp(NH)p-dependent activities displayed different sensitivities to thermal inactivation. Our results indicate that ?-adrenergic agonists and calmodulin interact with the same catalytic activity in cerebellar membranes, but presumably via two independent pathways.

Sarcoplasmic calcium-binding proteins in insect muscle Isolation and properties of locust calmodulin☆☆☆

No sarcoplasmic calcium-binding proteins reminiscent of those-described in other arthropods could be detected in locust flight and leg muscle. Instead, these tissue are rich in calmodulin. The purification and functional properties of this protein, which was purified to electrophoretic homogeneity, are very similar to those of calmodulin from bovine brain.

The activation of rabbit skeletal muscle phosphorylase kinase requires the binding of 3 Ca2+ per δ subunit

Abstract Rabbit skeletal muscle phosphorylase kinase (EC 2.7.1.38) binds 12 Ca 2+ , i.e. 3 per δ subunit; among these three Ca-binding sites, one has a high affinity (K diss = 0.31 μM) and two have a lower affinity (K diss = 2.15 μM). Thus, the binding of calcium to the enzyme-bound calmodulin, called δ subunit, is different from the binding to free calmodulin. The activation of phosphorylase kinase occurs when three Ca 2+ are bound to the δ subunit.

Regulatory proteins of crayfish tail muscle

Abstract Native tropomyosin and fragmented sarcoplasmic reticulum from crayfish tail muscle have been obtained in a pure state. After isoelectric precipitation, the former system yields two biologically active components, troponin and tropomyosin. Troponin binds calcium and can dissociate into three subunits of molecular weight 54 000, 29 000 and 16 000. The tropomyosin monomer has a molecular weight of 40 000. Desensitized actomyosin becomes calcium sensitive in the presence of native tropomyosin or tropomyosin plus troponin. The properties of crayfish fragmented sarcoplasmic reticulum (ATPase activity, calcium binding and uptake, inhibition of actomyosin superprecipitation) are characteristic of a calcium pump. Comparison with the regulatory system of rabbit skeletal muscle reveals many similarities between crayfish and mammalians; indeed crayfish fragmented sarcoplasmic reticulum or native tropomyosin can influence the calcium sensitivity of rabbit actomyosin.