The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1. Both the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca(2+) in the micromolar range. We have therefore compared the mechanisms and affinities for Ca(2+) activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca(2+)-pump ATPase was activated by Ca(2+) in a highly co-operative manner, with half-maximal activation occurring at about 0.3mum-Ca(2+). At an optimal Ca(2+) concentration, calmodulin stimulated the Ca(2+)-sensitive ATPase activity about 10-fold. 3. Ca(2+) activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca(2+) requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca(2+) activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg(2+), half-maximal activation of the phosphodiesterase was at about 3mum-Ca(2+). The presence of 1mm-Mg(2+) shifted the Ca(2+) activation curve to the right, as did elevation of the ionic strength. When the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg(2+) concentration, the ATPase was fully activated at 3mum-Ca(2+), whereas no polyphosphoinositide phosphodiesterase activity was detected below 100mum-Ca(2+). 5. The Ca(2+)-pump ATPase of the erythrocyte membrane normally maintains the Ca(2+) concentration of healthy erythrocytes below approx. 0.1mum. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.     

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.     

Lytic enzyme from lysates of Streptomyces venezuelae infected with actinophage MSP2

A lytic enzyme was purified 600-fold with 12% recovery from lysates of Streptomyces venezuelae S13 infected with actinophage MSP2. The purified enzyme preparation was homogeneous as shown by polyacrylamide electrophoresis. The enzyme was active over a pH range 6.0 to 9.0 with a maximum at pH 7.5. The pH profile for stability was sharp, with an optimum at pH 7.5. Maximal activity occurred between 30 and 35 C. The enzyme was stable at 20 C or less. A 30-min exposure to 25, 30, 35, 40, 45, and 50 C produced an inactivation of 3, 40, 77, 82, 93, and 100%, respectively. Lytic activity was stimulated fivefold by either 5 x 10(-3)m Mg(2+) or Mn(2+) and three- and twofold by Ca(2+) and Ba(2+), respectively. Addition of Na(+), K(+), NH(4) (+), or Li(+) to the tris(hydroxymethyl)aminomethane-hydrochloride buffer did not alter the rate of lysis. Enzyme activity was inhibited 74 and 27% by 10(-4) and 10(-5)m ethylenediaminetetraacetic acid (EDTA), respectively. The inhibition by EDTA was reversed partially by addition of Mg(2+). Lytic activity was abolished by either 5 x 10(-4)m HgCl(2) or p-hydroxymercuribenzoate, whereas 5 x 10(-4)m CuSO(4) inhibited 72%. Cell wall solubilization paralleled the release of N-terminal amino groups and reached a level of 0.23 mumole per mg of cell walls. No release of reducing power was detected in treated or untreated cell wall suspensions. Tests for proteolytic activity were negative.     

Calcium requirement for efficient phagocytosis by Dictyostelium discoideum

Extracellular EDTA suppressed in a dose-dependent manner the phagocytosis of yeast particles by Dictyostelium discoideum cells. Activity was restored fully by the addition of Ca(2+), and partially by the addition of Mn(2+)or Zn(2+), but Mg(2+)was ineffective. The pH-sensitive, Ca(2+)-specific chelator EGTA also inhibited phagocytosis at pH 7.5, but not at pH 5, and Ca(2+)restored the inhibited phagocytosis. In contrast, pinocytosis was unaffected by EDTA. Consistent with the idea that Ca(2+)was required for phagocytosis, D. discoideum growth on bacteria was inhibited by EDTA, which was then restored by the addition of Ca(2+). It is concluded that Ca(2+)was needed for efficient phagocytosis by D. discoideum amoebae. A search for Ca(2+)-dependent membrane proteins enriched in phagosomes revealed the presence of p24, a Ca(2+)-dependent cell-cell adhesion molecule-1 (DdCAD-1) that could be the target of the observed EDTA and EGTA inhibition. DdCAD-1-minus cells, however, had normal phagocytic activity. Furthermore, phagocytosis was inhibited by EDTA and rescued by Ca(2+)in the mutant just as in wild type. Thus, DdCAD-1 was not responsible for the observed Ca(2+)-dependence of phagocytosis, indicating that one or more different Ca(2+)-dependent molecule(s) was involved in the process.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

A comparison of methods for the isolation and fractionation of reticulocyte ribosomes

1. Polysomes, ribosomes and pH5 enzymes were isolated from rabbit reticulocytes by acidifying the post-mitochondrial supernatant to pH6.0 to precipitate all ribonucleoprotein particles and about half the pH5 enzymes; the precipitate was redissolved in buffer, pH7.6, and fractionated by zone centrifuging. 2. The isolation of polysome-rich and ribosome-rich fractions from the post-mitochondrial supernatant was also examined. 3. Studies of the stability of polysomes revealed that dissociation into sub-units occurred when both bound and free Mg(2+) was chelated by EDTA or when the pH was increased above pH8.8.     

Purification and characterization of phosphoenolpyruvate carboxylase from Plasmodium berghei

Phosphoenolpyruvate (PEP) carboxylase was purified over 400-fold from Plasmodium berghei. The purified enzyme was stable in 0.4 m potassium phosphate buffer (pH 7.4) containing 0.5 m glucose, 1 mm ethylenediaminetetraacetic acid (EDTA), and 1 mm MgCl(2). It had a molecular weight of 280,000 determined by sucrose density gradient centrifugation in this buffer, but it aggregated and was unstable in the presence of different salts or a more dilute solution of potassium phosphate. The K(m) for PEP was 2.6 mm and that for Mg(2+) was 1.3 mm. The K(m) for bicarbonate was 2 mm. Citrate, nucleotides, and EDTA inhibited the PEP carboxylase of P. berghei by decreasing the concentration of free magnesium ions, but acetyl-coenzyme A, fructose-1,6-diphosphate, and aspartate did not influence its activity. A chloroquine concentration of 1.8 x 10(-4)m inhibited the enzyme 50%.     

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.     

Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.     

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system.

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.     

Characterization of calcium-ion-activated adenosine triphosphatase in the plasma membrane of rat mast cells.

The properties of a Ca2+ activated adenosine triphosphatase shown to be present in homogenates of purified rat peritoneal mast cells were investigated. The enzyme was activated by Ca2+, Mg2+, and to a lesser extent by Mn2+ and Co2+. Ca2+ alone was necessary for full activity and the further addition of Mg2+ did not have any effect. The chelating agents EGTA (ethanedioxybis(ethylamine)tetra-acetate) and EDTA completely inhibited the reaction. The pH optimum was 7.8. Reduced glutathione, cysteine, dithiothreitol, N-ethylmaleimide, urea, ADP, NaF, increasing ionic strength and Triton X-100 all inhibited the reaction. On subcellular fractionation of mast-cell homogenates by density-gradient centrifugation, the distribution of Ca2+ activated adenosine triphosphatase resembled that of 5'-nucleotidase, but differed from that of the other markers used, suggesting localization in the plasma membrane. Further experiments indicated that the enzyme is present on the external surface of the plasma membrane.     

Membrane adenosine triphosphatase of Micrococcus lysodeikticus: effect of millimolar Mg2+ in modulating the properties of the membrane-bound enzyme

The latency of Micrococcus lysodeikticus membrane-bound Mg(2+)-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg(2+) (50 mM, 10 mM, or none) and by washing them with different Mg(2+)- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg(2+) bound to at least two classes of sites. The binding of Mg(2+) to low-affinity sites (saturation at approximately 40 mM external Mg(2+)) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg(2+)) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg(2+) (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg(2+)/Ca(2+) antagonism as activators of the membrane ATPase was not directly related to Mg(2+) binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg(2+). Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca(2+)-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg(2+)-ATPase-membrane complex.     

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.     

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.     

Comparison of nuclear 5 alpha-reductase activities in the stromal and epithelial fractions of human prostatic tissue

The nuclear conversion of testosterone (T) to dihydrotestosterone (DHT) was compared in the separated stromal and epithelial fractions of hyperplastic (n = 20), malignant (n = 5) and normal (n = 1) prostatic tissues. Standard assay conditions were: 1 microM testosterone, plus 4-6 X 10(5) DPM [3H]T, 1.0 mM NADPH, 2.0 mM EDTA and 0.5-1.0 mg nuclear protein in a total volume of 1.1 ml HEPES buffer, pH 7.4 (stroma) or MES buffer, pH 6.5 (epithelium). The apparent Km values for the stromal enzyme were 0.2, 0.2 and 0.3 microM, respectively, for the enzymes in hyperplastic, malignant and normal tissues. The Vmax values were 26 +/- 4.2, 2.8 +/- 0.6 and 4.1 pmol/mg protein/30 min incubation, respectively, for these same tissues. The apparent Km values for the epithelial enzymes, from the same tissues, were 0.03, 0.07 and 0.08 microM. The Vmax values for the epithelial enzymes were 4.8 +/- 1.2, 0.69 +/- 0.08 and 1.1 pmol/mg protein/30 min incubation. The pH optimum for the stromal enzyme lay between pH 6.5 and 7.5, whereas the pH optimum for the epithelial enzyme lay between 5.5 and 6.5. Enzymatic activity in both fractions revealed a biphasic response to zinc. In the absence of EDTA, microM quantities of zinc enhanced enzymatic activity while mM quantities inhibited this activity. These results would suggest that differences in the conversion of T to DHT help to explain, at least in part, the higher DHT levels seen in hyperplastic tissue and the higher T levels seen in the malignant prostate.     

Purification and general properties of δ-aminolaevulate dehydratase from Nicotiana tabacum L

1. delta-Aminolaevulate dehydratase (EC 4.2.1.24) was purified 80-fold from tobacco leaves and its properties were studied. 2. The enzyme had optimum pH7.4 in potassium phosphate buffer, K(m)6.25x10(-4)m at 37 degrees and pH7.4, optimum temperature 45 degrees and an activation energy of 11100 cal./mole. 3. The enzyme lost activity when prepared in the absence of cysteine, and this activity was only partly restored by the later addition of thiols. Reagents for thiol groups inactivated the enzyme. 4. Mg(2+) was essential for activity, and EDTA and Fe(2+) were inhibitory; Mn(2+) was an activator or an inhibitor depending on the concentration.     

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. I. General properties of variously prepared membranes and the mechanism of the isosmotic imidazole effect.

1. Membranes prepared from human erythrocytes hemolyzed in isosmotic (310 imosM) imidazole buffer, pH 7.4, show enhanced and stabilized (Ca2+ + Mg2+)-ATPase activity compared with membranes prepared from erythrocytes hemolyzed in hypotonic (20 imosM) phosphate or imidazole buffer, pH 7.4. 2. Exposure of intact erythrocytes or well-washed erythrocyte membranes to isosmotic imidazole does not cause enhanced (Ca2+ + Mg2+)-ATPase activity. 3. Exposure of erythrocyte membranes, in the presence of isosmotic imidazole, to the supernatant of erythrocyte hemolysis or to a partially purified endogenous (Ca2+ + Mg2+)-ATPase activator, promotes enhanced (Ca2+ + Mg2+)-ATPase activity. Under appropriate conditions, NaCl can be shown to substitute for imidazole. The results demonstrate that imidazole does not act directly on the erythrocyte membrane but rather by promoting interaction between an endogenous (Ca2+ + Mg2+)-ATPase activator and the erythrocyte membrane.     

Intracellular localization of dinucleosideoligophosphate phosphorylase from Euglena gracilis

1. Formation of labeled ADP in the ADP-(32)P -exchange reaction, the inherent property of the dinucleosideoligophosphate (DNOP) phosphorylase demonstrated recently in Euglena gracilis can be used to detect specifically the enzyme activity in crude extracts among interfering enzymes like phosphodiesterase I, adenylate kinase or non-specific phosphatases. 2. The initial velocity was measured in an incubation mixture lacking Mg(2+) but containing EDTA and/or Ap(5)A an inhibitor of the adenylate kinase. 3. Using different sucrose-density gradients and controlling activities of the marker enzymes it has been proved that the DNOP phosphorylase is a cytosolic enzyme in E. gracilis.