Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the 'microsomes' (13000-80000 X g sediment) from pea stem tissue is strongly influenced by concentration of Mg2+ in the homogenization medium. The absence of Mg2+ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at 80000 X g upon addition of 5-10 mM Mg2+ (or Mn2+, Ca2+, Zn2+) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl2 the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg2+. Microsomes prepared with Mg2+ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H⁺ fluxes in pea stem microsomal vesicles. The H⁺/K⁺ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H⁺ translocation. The ATP-dependent acridine orange uptake is Mg²⁺-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na₃VO₄. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.     

Temperature-dependent relationship between K+ influx,Mg2+-ATPase activity,transmembrane potential and membrane lipid composition in mycoplasma

The temperature-dependent relationship between K⁺ active influx, Mg²⁺-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of ⁴²K⁺ active influx gave a linear relationship for (chol (+), O + P) cells ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?9}\text{kcal}$). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ($\text{t > 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?24}\text{kcal}\text{; t < 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?40}\text{kcal}$).Finally, a biphasic response with a break at approx. 23°C ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?7}\text{kcal}\text{, t > 23°}\text{C}$; $\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?44}\text{kcal}\text{, t < 23°}\text{C}$) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K⁺ influx and the temperature dependence of both the Mg²⁺-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K⁺ transport at the level of the K⁺ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K⁺ carrier is associated with the most fluid lipid species present in the membrane.     

Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity

Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker ([¹⁴C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100–200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets.     

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex: Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na⁺ + K⁺)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na⁺ + K⁺)-ATPase activity and [³H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na⁺ + K⁺)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na⁺ and K⁺. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN^?, is strongly hyperpolarized when ATP is present in the incubation medium.The stimulation of SCN^? uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN^? uptake is strongly inhibited by ATPase inhibitors such as $\text{p-}\text{chloromercuribenzenesulphonate}$ and $\text{N,N}{}^{\mathrm{\prime }}\text{-}\text{dicyclohexylcarbodiimide}$ and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN^? uptake. The proton conductor carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ strongly inhibits ATP-stimulated SCN^? uptake. The effect of ATP on SCN^? uptake depends on the pH of the medium, the maximum being reached at about pH 7.0.These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H⁺.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Modulation of c-myc expression in the HL-60 cell line

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.     

Overproduction and purification of initiation factor IF2 and pNUSA proteins from a recombinant plasmid bearing strain

The genes for translational initiation factor, IF2 and pNusA have been cloned into a plasmid vector where they are placed under the control of the inducible lambdapL promoter and the c1857 thermosensitive repressor. When a strain carrying this plasmid is heat induced, IF2 alpha, IF2 beta and pNusA are overproduced 15 to 20 fold. This has allowed us to purify the IF2 and NusA proteins in large amounts.     

Levels of collagenolytic activity, β-glucuronidase,and collagen prolyl hydroxylase in paws from rats with developing adjuvant arthritis

The collagenolytic activity associated with insoluble collagen fibers separated from homogenates of inflamed paws from rats with adjuvant arthritis was quantitated using EDTA-sensitive solubilization of hydroxyproline as a measure of activity. Approximately 60% of the solubilized hydroxyproline was associated with dialyzable products. The level of collagenolytic activity in the paws increased with time after the induction of adjuvant arthritis and paralleled to a large extent the development of inflammation in both the adjuvant injected (right) hind paw and in the non-injected, contralateral paw. By day 26, the level of free collagenolytic activity in the injected paw had increased to a level 30 times normal while that in the contralateral paw had increased to a level 10 times normal. Treatment of the residues from the injected paws with trypsin resulted in the activation of a latent collagenolytic activity which, on day 26, accounted for approximately 50% of the total activity. The elevated level of collagen prolyl hydroxylase in the inflamed paw suggested that the rate of collagen synthesis was also increased. The activity of β-glucuronidase increased in the inflamed paw with time after the induction of adjuvant arthritis while that of cathepsin G was elevated as compared to normal in paws removed, 5 but not 22 days after the induction of adjuvant arthritis. The inflamed paw of the adjuvant rat may represent a useful system in which to study the role of collagenolytic enzymes in the destruction of connective tissue by inflammatory lesions.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Changes in activity of enzymes associated with prespore differentiation in a suspension culture of Dictyostelium discoideum

It has been shown (Okamoto, K. (1981) J. Gen. Microbiol. in the press) that Dictyostelim discoideum cells dissociated at early aggregation can differentiate into prespore cells in a suspension containing glucose, albumin, EDTA and cyclic AMP. Strict requirement of cyclic AMP in this process has also been demonstrated. In the present paper, changes in activity of eight developmentally regulated enzymes were examined in this culture system and compared to those occuring in the normal course of development on the solid substratum. The results show that (a) formation in this medium is not accompanied by increases in activity of UDPglucose pyrophosphorylase and trehalose phosphate synthetase, unlike the case of the normal development, (b) among the enzymes examined, only UDPgalactose: polysaccharide galactosyl transferase can be regarded as a specific marker of the prespore formation, and (c) development in this system does not proceed beyond the slug stage of the normal development, in the case of a wild-type strain NC4.     

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland

The stimulatory effect of spermidine on the translation of poly(A)⁺ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg²⁺ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Protein kinase activity on the cell surface of a macrophage-like cell line,J774.1 cells

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [γ-³²P]ATP, a transfer of [³²P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg²⁺-dependent but cAMP-independent, and Mg²⁺ could be substituted for by Mn²⁺. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonoester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.