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.     

Structural reorganisation of chloroplast thylakoid membranes in response to heat-stress

Chloroplasts isolated from broad bean (Vicia faba) show major structural reorganisations on heating to temperatures above 35°C. Exposure to increasing temperatures in the range 35–45°;C for 5 min, leads to a progressive destacking of the chloroplast membranes and the replacement of the normal granal arrangement by modified thylakoid attachment sites. An analysis of the size and packing densities of the freeze-fracture particles present in different membrane fracture-faces suggests that this rearrangement reflects the dissociation of the light-harvesting units of Photosystem II. The antennae complexes of Photosystem II appear to cluster together, maintaining regions of membrane adhesion, whilst excluding the core-complexes of Photosystem II and light-harvesting units of Photosystem I from these regions. If the chloroplasts are heated to higher temperatures, 45–55°C, phase-separated aggregates of non-bilayer-forming lipids are often observed. The release of these lipids from their normal constraints within the bilayer is consistent with the idea that they play a role in the packaging of the light-harvesting complexes within the thylakoid membrane.     

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H⁺ ejection/2e^? ratio approaches an average value of 3 when K⁺ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ejection/2e^? ratio of 2 is observed. (3) The low stoichiometry of 3H⁺ ejected (instead of 4) per 2e^? and the high rate of H⁺ back-decay (0.1615 $\text{ln}\text{δ-}\text{(}\text{ngatom}\text{)}\text{H}{}^{\mathrm{+}}\text{s}$ and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K⁺/H⁺ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.     

A possible rate-limiting function of chloroplast hexosemonophosphate isomerase in starch synthesis of leaves

Levels of fructose 6-phosphate and glucose 6-phosphate were measured in chloroplasts which had been isolated non-aqueously from leaves of various plants. a large decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate in the light indicated considerable displacement of the hexosephosphate isomerase reaction from equilibrium in leaves of spinach and red beet which were photosynthesizing at high rates. The decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate was correlated with an increase in the chloroplastic level of 3-phosphoglyceric acid, which proved to be a competitive inhibitor of chloroplast hexosephosphate isomerase. Other metabolites, especially the product of the reaction, glucose 6-phosphate, and ions in concentrations as present in the stroma under natural conditions, cause a further reduction in the rate of the forward reaction of the hexosemonophosphate isomerase. When the concentration of O₂ in air was decreased from 21 to 2%, both the rate of leaf photosynthesis and the ratio of glucose 6-phosphate to fructose 6-phosphate increased, whereas the concentration of 3-phosphoglyceric acid and starch synthesis decreased. The results are explained in terms of activation of ADPglucose pyrophosphorylase and of inhibition of hexosephosphate isomerase by 3-phosphoglyceric acid. Hexosephosphate isomerase appears to assume a rate-limiting function in starch synthesis in the light when ADPglucose pyrophosphorylase is activated.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Generation of phosphodiesters during fast-to-slow muscle transformation A 31P-NMR study

During rabbit fast-to-slow twitch muscle transformation, in response to electrical stimulation, the compound glycerophosphocholine can be detected in these muscles by ³¹P-NMR. This compound is not detectable in contralateral control muscles but is present in slow twitch soleus.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts

An average target size of 251 kDa has been obtained for the (Ca²⁺ + Mg²⁺)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer.     

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts

Intact spinach chloroplasts, washed four times in buffered sorbitol to decrease catalase contamination, supported O₂ evolution in the dark at very low rates (less than 2 μmol/mg Chl per h) in the presence of low concentrations of H₂O₂ (0.25 mM); H₂O₂ was not significantly metabolished under these conditions. In the light, washed chloroplasts supported H₂O₂-dependent O₂ evolution at rates of 28–46 μmol/mg Chl per h in the presence of 0.1–0.25 mM H₂O₂; the concentration of H₂O₂ supporting 0.5V_max was estimated to be 25 μM. O₂ evolution in the light was associated with H₂O₂ consumption and ceased after the production of 0.45 mol per mol H₂O₂ consumed. Both O₂ evolution and H₂O₂ consumption were abolished by 5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Washed intact chloroplasts contained endogenous pools of GSH and ascorbate estimated at 10 and 33 mM, respectively. H₂O₂-dependent O₂ evolution in the light was associated with a decrease in these levels which increased as O₂ evolution gradually ceased. The results are consistent with the hypothesis that H₂O serves as eventual electron donor for the reduction of H₂O₂ in illuminated chloroplasts and that GSH/GSSG and ascorbate/dehydroascorbate serve as intermediate electron carriers. Preincubation of chloroplasts in the dark with 0.1 mM H₂O₂ abolished O₂ evolution in the light.     

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [³⁵S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

Assay of interferon-mediated protein kinase activity from plasma and tissue extracts

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous 67,000 and 72,000 Mr proteins, respectively. Enhanced levels of such kinase activity are also detectable in the plasma of patients treated with interferon and in the plasma and tissues of interferon-treated mice. A rapid and efficient method of assay for these protein kinase activities is described. The samples are first incubated with heparin (100 units/ml), which results in the inhibition of different protein kinase activities, but not the one mediated by interferon. The latter one is then assayed after partial purification on poly(rI):(rC)-Sepharose or poly(rG)-Sepharose. The protein kinase from human and mouse cells in culture and from the different tissues of mice binds specifically to poly(rI):(rC)-Sepharose. On the other hand, the protein kinase activity from both mouse and human plasma shows a higher affinity toward poly(rG)-Sepharose. These methods are successfully applied for the determination of the interferon-mediated protein kinase activity from tissue extracts and plasma.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Na+-Ca2+ exchange in the axolemma-rich membrane vesicle preparations from the walking-leg nerves of the american lobster

An axolemma-rich membrane vesicle fraction was prepared from the leg nerve of the lobster, Homerus americanus. In this preparation Ca²⁺ transport across the membrane was shown to require a Na⁺ gradient (Na⁺-Ca²⁺ exchange), and external K⁺ was found to facilitate this Na⁺-Ca²⁺ exchange activity. In addition, at high Ca²⁺ concentrations (20 mM) a Ca²⁺-Ca²⁺ exchange system was shown to operate, which is stimulated by Li⁺. The Na⁺-Ca²⁺ exchange system is capable of operating in the reverse direction, with Ca²⁺ uptake coupled with Na⁺ efflux. Such a vesicular preparation has the potential for providing useful experimental approaches to study the mechanism of this important Ca²⁺ extrusion system in the nervous system.     

Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration

An increase in cytoplasmic free [Ca²⁺], [Ca²⁺]_i, has been suggested as the trigger for the permeability changes that bring about cell volume restoration following exposure to anisotonic media. This idea was directly tested in human peripheral lymphocytes undergoing regulatory volume decrease following a hypotonic dilution of the suspension. [Ca²⁺]_i was measured with the intracellularly trapped fluorescent indicator, quin2, and showed no measurable change on hypotonic swelling or during the subsequent volume decrease. Moreover, even though the incorporated quin2 adds significant Ca-buffering to the cytoplasm, regulatory volume decrease occurred normally in the quin2-loaded cells. It appears that alterations in [Ca²⁺]_i are not involved in these processes of volume regulation. An intracellularly trapped derivative of fluorescein, bis(carboxyethyl)carboxyfluorescein, was used to monitor cytoplasmic pH, which also showed no change during regulatory volume decrease.     

Is cytoplasmic Ca2+ in lymphocytes elevated in cystic fibrosis

An increased cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca²⁺]_i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca²⁺]_i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca²⁺ stores were also comparable in both types of individual.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.