Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major M_r = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a M_r = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca²⁺-stimulated protease. Neonatal lung extracts show more Ca²⁺-stimulated proteolysis of R subunits than adult extracts, although only slight agerelated differences in either the Ca²⁺-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N₃-[³²P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (R_I) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca²⁺-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.     

Stimulation of mouse liver corticosteroid side chain isomerase by cobaltous and nickelous ions: evidence for an endogenous inhibitor of isomerase activity

Corticosteroid side chain isomerase of mouse liver cytosol was stimulated by Co2+ and Ni2+. The magnitude of stimulation increased with incubation time. For Co2+ and Ni2+, respective enhancements were 2.8- and 4.0-fold at 15 min and 3.9- and 5.0-fold at 60 min. The relationship between steroid substrate concentration (11-deoxy-[21-3H]corticosterone) and initial velocity was consistent with a model in which the cations reacted with a cytosol inhibitor of isomerase activity. Enzyme, partially purified by ammonium sulfate fractionation and gel filtration, had a 6.8-fold increased specific activity. Co2+ and Ni2+ enhanced the activity of partially purified enzyme 1.6- and 1.9-fold. Unlike the cytosol, stimulation was achieved without lag and was not altered by prolonged incubation. Metal ion chelating agents did not have a consistent effect on the activity of the partially purified enzyme. Cyanide and alpha,alpha-dipyridyl increased, and dithizone and 8-hydroxyquinoline decreased activity. The data are not consistent with the hypothesis that side chain isomerase is a metalloenzyme. It is concluded that Co2+ and Ni2+ stimulate the enzyme by removing an endogenous inhibitor.     

Ca2+-ATPase from sarcoplasmic reticulum: acylphosphatase activity

Fractionation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle was performed by solubilization of the vesicles in the presence of deoxycholate, followed by sucrose density gradient centrifugation and gel filtration chromatography. This procedure permitted the isolation of essentially pure Ca²⁺-ATPase; this enzyme showed ATPase as well as acylphosphatase activity, both activities being clearly enhanced by deoxycholate. The acylphosphatase activity of the purified Ca²⁺-ATPase was characterized with regard to some kinetic properties, such as pH, Mg²⁺, Ca²⁺, and deoxycholate dependence, and substrate affinity, determined in the presence of acetylphosphate, succinylphosphate, carbamylphosphate, and benzoylphosphate; in addition, the stability of both activities was checked in time-course experiments. The main similarities between the two activities, such as the Mg²⁺ requirement, the deoxycholate activation, and the pH dependence, together with the competitive inhibition of the benzoylphosphatase activity by ATP, the inhibition of both activities by tris(bathophenanthroline)-Fe²⁺, and the relief of this inhibitory effect by carbonylcyanide-4-trifluoromethoxyphenyl hydrazone support the hypothesis that acylphosphatase and ATPase activities of sarcoplasmic reticulum vesicles reside in the same active site of the enzyme. With regard to possible relationships between acylphosphatase activity of the purified Ca²⁺-ATPase and “soluble” acylphosphatase present in the 100,000g supernatant fraction, comparison of some kinetic and structural parameters indicate that these two activities are supported by quite different enzymes.     

Hydrolysis by horse muscle acylphosphatase of (Ca2+ + Mg2+)-ATPase phosphorylated intermediate

Horse muscle acylphosphatase (EC 3.6.1.7) was found to hydrolyze the labeled phosphorylated intermediate of (Ca²⁺ + Mg²⁺)-ATPase from rabbit muscle. In addition, the phosphorylated peptides obtained by pepsin digestion of the labeled phosphorylated microsomes were completely hydrolyzed by acylphosphatase. These findings suggest a possible regulatory role of this enzyme in vivo on the calcium transport process by sarcoplasmic reticulum.     

Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.     

Regulation by Mg2+ and Ca2+ of mitochondrial membrane integrity: study of the effects of a cytosolic molecule and Ca2+ antagonists.

The coupling of aliphatic amines to agarose by the cyanogen bromide reaction yields isourea linkages which are positively charged at pH 7. The presence of these cationic sites in affinity gels causes significant non-specific adsorption of proteins. Serum albumin was found to bind to a number of derivatized gels which possessed these charged groups. The use of adipic dihydrazide as the leash moiety yielded affinity gels which were noncharged at pH 7. Serum albumin failed to adsorb to these gels. Beta-galactosidase from Escherichia coli was found to be sensitive to both ionic and hydrophobic groups in an affinity gel. A sample of active-site inhibited enzyme was found to bind to an affinity gel which contained both the cationic isourea and a phenyl structure in the leash. Thus it was concluded that the affinity purification of this enzyme has yet to be demonstrated. These studies dictate against the use of salt and pH gradients to desorb enzymes from affinity sorbents.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

Multiple specificities of brain Ca2+- and calmodulin-dependent protein kinase for substrate

The purified Ca2+- and calmodulin-dependent protein kinase from rat brain, which has a M.W. of 120,000 by gel filtration analysis, showed a broad substrate specificity. In addition to myosin light chain from chicken gizzard, the enzyme phosphorylated myelin basic protein, casein and two endogenous substrates in a Ca2+- and calmodulin-dependent manner. In contrast, chicken gizzard myosin light chain kinase exclusively phosphorylated myosin light chain.     

Lectin- and ionophore-stimulated Ca2+ influx in murine lymphocytes: inhibition by disialoganglioside

Gangliosides suppress lymphocyte mitogenesis when added exogenously to the cells. On the premise that the mechanism of ganglioside action may be an interference with primary induction events, mitogen-induced 45Ca2+ influx in murine lymphocytes was studied. Disialoganglioside (GD1a) at physiopathological concentrations inhibits concanavalin A-induced 45Ca2+ uptake as well as blast transformation. The suppressive action of GD1a is both concentration dependent (50% suppression at 13 microM) and very rapid (within 1 min). GD1a is not cytotoxic nor does it significantly alter the rate of Ca2+ efflux. The uptake studies were extended to A23187, a compound with mitogenic and specific divalent cation ionophore activities. Ca2+ uptake by lymphoid cells from AKR/J, Swiss, and CBA mice is stimulated by A23187; and GD1a, in a dose-dependent manner, inhibits the ionophore-induced 45Ca2+ influx. Pretreatment of thymocytes with GD1a renders the cells greatly insensitive to the subsequent ionophore activity of A23187. The results suggest that exogenous gangliosides may function as an inhibitor of some of the mitogen-triggered early events, including Ca2+ metabolism, and thus influence the immunological behavior of intact lymphoid cells.     

Regulation of the betaine content of rat liver

The dietary levels of both choline and protein are major determinants of the content of betaine in rat liver. Increased protein intake decreases hepatic betaine. Our studies indicate that an increase in the betaine-homocysteine methyltransferase reaction due to an increased availability of homocysteine is the basis for this effect of dietary protein.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

The effect of phenothiazines on Ca2+ fluxes in skeletal muscle sarcoplasmic reticulum

The effect of phenothiazines (trifluoperazine, chlorpromazine, methochlorpromazine, and imipramine) on Ca2+ fluxes in light and heavy sarcoplasmic reticulum (SR) isolated from rabbit fast-twitch skeletal muscle was investigated. These drugs inhibited Ca2+ loading and (Ca2+,Mg2+)-ATPase activity, but had no effect on unidirectional Ca2+ efflux from vesicles loaded either actively or passively with Ca2+. Chlorpromazine, which is membrane permeable, and its quaternary analog, methochlorpromazine, which is membrane impermeable, gave identical results. It is concluded that (a) the enhancement of net Ca2+ release by phenothiazines is due to inhibition of Ca2+ influx mediated by the Ca2+ pump rather than to the opening of a Ca2+ channel; and (b) phenothiazines act at the outer (myoplasmic) face of the SR membrane.     

Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypanosoma brucei requires Ca2+

A-antigenicity; Glycocalyx; Hydrolases; Membrane glycoproteins     

Glycerolipid biosynthesis in rat adipose tissue 12. Properties of Mg2+-dependent and -independent phosphatidate phosphohydrolase

The properties of Mg²⁺-dependent and Mg²⁺-independent phosphatidate phosphohydrolase activities were investigated in different subcellular fractions in rat adipose tissue. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. The Mg²⁺-dependent phosphatidate phosphohydrolase was inhibited in the presence of N-methyl- or N-ethylmaleimide, whereas the Mg²⁺-independent activity was unaffected by these agents. The Mg²⁺-dependent phosphatidate phosphohydrolase was more sensitive to proteolysis and to high temperature (55 °C) compared to the Mg²⁺-independent enzyme. The Mg²⁺-dependent phosphatidate phosphohydrolase activity was reduced significantly during aging without any appreciable effects on the Mg²⁺-independent phosphatidate phosphohydrolase activity. These studies demonstrate that, in addition to Mg²⁺-dependency, these two forms of phosphatidate phosphohydrolases differ in several respects irrespective of their location in the adipose cell.