Cytosolic calcium dependent proteinase of human erythrocytes: Formation of an enzyme-natural inhibitor complex induced by Ca2+ ions

A calcium dependent soluble neutral proteinase has been purified to homogeneity from human erythrocytes. The proteinase is composed of two different polypeptide chains of approximate molecular weight of 80 k and 30 k daltons. Maximum activity is expressed at 50 μM Ca²⁺. The enzyme is regulated by reversible binding to a natural inhibitor, also present in the cytosolic compartment. The formation of the enzyme-inhibitor complex is dependent on high Ca²⁺ concentrations and is reversed by chelating agents. The proteinase is inhibited by leupeptin, chymostatin, antipain and free hemin and has a marked specificity for native or denatured human globin chains.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Some kinetic properties of phosphorylated ATPase of sarcoplasmic reticulum formed in the absence of added alkali metal salts.

In the rat both hypothyroidism and diabetes decrease heparin-releasable liver lipase activity. This defect may be reversed by feeding a diet rich in polyunsaturated fatty acids. It is suggested that a diet-induced increase of membrane fluidity restores liver lipase activity, which contributes to the hypolipidemic effect of polyunsaturated fatty acids.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.     

On the existence of two populations of mitochondria in a single organ. Respiration, calcium transport and enzyme activities.

Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome $\text{c}$ to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca²⁺ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome $\text{c}$ content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.     

Effects of chelating agents on the initial interaction of phytohemagglutinin with lymphocytes and the subsequent stimulation of amino acid uptake

The chelating agents, ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) and EDTA, had no effect on the initial interaction of phytohemagglutinin with lymphocytes at concentrations which have been shown previously to inhibit the development of the phytohemagglutinin response completely. However, they had a marked inhibitory effect on uptake of the amino acid analog, α-aminoisobutyric acid in both unstimulated and phytohemagglutinin-stimulated cells. The inhibition of amino acid uptake by EGTA could be reversed by adding Ca²⁺ but not Mg²⁺. These results demonstrated that Ca²⁺ is not essential to the initial interaction of phytohemagglutinin with the cell, but does influence amino acid transport which may be a critical preparatory event for later increased protein synthesis.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Cation dependent conformations of brain CA2+-dependent regulator protein detected by nuclear magnetic resonance.

The 250MHz NMR spectrum of the brain Ca²⁺-dependent regulator protein was examined in the absence of cations and in the presence of Ca²⁺ or Mg²⁺. The Ca²⁺-saturated regulator protein and Mg²⁺-saturated regulator protein exhibited several spectral differences in the aromatic and aliphatic regions of their spectra. Certain spectral changes observed to occur upon addition of metal ions are qualitatively similar to those which have been observed in the spectrum of skeletal troponin-C. These results suggest that the large sequence homology between skeletal troponin-C and the regulator protein results in similar conformational changes due to the binding of Ca²⁺ or Mg²⁺.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

The effect of oncomodulin on cAMP phosphodiesterase activity

Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43, 5390-5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.     

Na+, K+-ATPase: evidence for the binding of ATP to the phosphoenzyme

Highly purified Na⁺, K⁺-ATPase of the dog kidney was reacted with Mg²⁺+³²Pi or Mg²⁺+³²Pi + ouabain. ³²P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K_0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na⁺. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

Ca2+-dependent cyclic nucleotide phosphodiesterase from rabbit lung.

Studies are presented which demonstrate that rabbit lung contains both Ca²⁺-activated cyclic nucleotide phosphodiesterase and calmodulin activity. The Ca²⁺-activatable cyclic nucleotide phosphodiesterase is different from the common type in that it contains tightly bound calmodulin. The bound calmodulin is not dissociated from the enzyme even in very low concentrations of Ca²⁺ after DEAE-cellulose and Sephadex G-200 column chromatography.     

High-affinity calcium-binding proteins in Escherichia coli

Crude extracts of Escherichia coli contain at least three heat stable proteins of Mr, 33,000, 47,000, and 60,000, which bind 45Ca2+ in buffers containing micromolar calcium and physiological salt concentrations. Fractions containing these proteins neither activated the calmodulin-dependent enzyme, NAD kinase, nor inhibited the activity of this enzyme in the presence of brain calmodulin. Radioimmunoassay of crude extracts for calmodulin indicated the presence of a calmodulin-like antigen. Crude extracts also contain proteins that interact with 2-trifluoromethyl-10H-(3'-aminopropyl)phenothiazine-Sepharose in a calcium-dependent manner, but proteins eluted from this resin did not bind calcium with high affinity.