In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.     

In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates

Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.     

Activation of adenylate cyclase in Acanthamoeba palestinensis

Preincubation of Acanthamoeba palestinensis homogenates in 0.25M sucrose-TM (2mM MgSO4 and 5mM Tris-HCl, pH 7.4) at 0 degree C for increasing periods of time up to 3 h, leads to a progressive increase in the activity of adenylated cyclase. In contrast, preincubation of isolated membrane fractions enriched in enzyme activity in the same medium results in no activation. However, preincubation of membrane fractions in medium containing a high density of sugars (sucrose, glucose or fructose) mimics the activation obtained with homogenates. The high density sugar activation is time and temperature dependent, and reversible upon return to a low density medium. The high osmotic pressure of the sugars utilized may be a factor, since high concentrations of the sucrose polymer, Ficoll, which has low osmotic activity, causes not activation. Soluble activators, protein synthesis and changes in cyclic nucleotide phosphodiesterase activity were all eliminated as possible effectors of the apparent activation of adenylate cyclase. In contrast to mammalian adenylate cyclase, the endoplasmic reticulum localized enzyme of Acanthamoeba is inhibited by NaF and is unaffected by GTP, adenosine, epinephrine, prostaglandin E1, propranolol, and meclofenamic acid. These data indicate that the adenylate cyclase of Acanthamoeba is structurally different from that of most mammalian cells.     

Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB

The low-shear viscosity of 5-30 microM F-actin was greatly increased by the addition of 0.1-0.5 microM unphosphorylated Acanthamoeba myosins IA and IB. The increase in viscosity was about the same in 2 mM ADP as in the absence of free nucleotide but was much less in 2 mM ATP. The single-headed monomolecular Acanthamoeba myosins were as effective as an equal molar concentration of two-headed muscle heavy meromyosin and much more effective than single-headed muscle myosin subfragment-1. These results suggest that Acanthamoeba myosins IA and IB can cross-link actin filaments as proposed in the accompanying paper (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179) to explain the actin-dependent cooperative increase in actin-activated Mg2+-ATPase activity as a function of the concentration of myosin I. Superprecipitation occurred when phosphorylated myosin IA or IB was mixed with F-actin. In addition to myosin I heavy chain phosphorylation, superprecipitation required Mg2+ and ATP. ATP hydrolysis was linear during the time course of the superprecipitation, and inhibitors of ATP hydrolysis inhibited superprecipitation. A small, dense contracted gel was formed when the reaction was carried out in a cuvette, and a birefringent actomyosin thread resulted from superprecipitation in a microcapillary. The rate and extent of superprecipitation depended on the actin and myosin I concentrations with maximum superprecipitation occurring at an actin:myosin ratio of 7:1. These results provide strong evidence for the ability of Acanthamoeba myosins IA and IB to perform contractile and motile functions.     

Interactions of nucleotide analogues with rod outer segment guanylate cyclase.

Light activation of cyclic GMP hydrolysis in rod outer segments is mediated by a G-protein which is active in the GTP-bound form. Substitution of GTP with a nonhydrolyzable GTP analogue is thought to leave the G-protein in a persistently activated state, thereby prolonging the hydrolysis of cyclic GMP. Restoration of cyclic GMP concentration in the cell also depends upon GTP since it is the substrate for guanylate cyclase, but little is known about the effects of GTP analogues on this enzyme. We report here the effects of the analogues of GTP and ATP as inhibitors and substrates of rod disk membrane guanylate cyclase. The rate of cyclic GMP synthesis from GTP in rod disk membranes was about 50 pmol min-1 (nmol of rhodopsin)-1. Analogues of GTP and adenine nucleotides competitively inhibited the cyclase activity. The order of inhibition, with magnesium as metal cofactor, was ATP greater than GMP-PNP greater than AMP-PNP approximately GTP-gamma-S; with manganese, AMP-PNP was more inhibitory than GTP-gamma-S. The inhibition constants, with magnesium as cofactor, were 0.65-2.0 mM for GTP-gamma-S, 0.4-0.8 mM for GMP-PNP, 1.5-2.3 mM for AMP-PNP, and 0.07-0.2 mM for ATP. The fraction of cyclase activity inhibited by analogues was similar at 1 and 0.03 microM calcium. Besides inhibition of cyclase, the analogues also served as its substrates. GTP-gamma-S substituted GTP with about 85% efficiency while GMP-PNP and ATP were about 5 and 7% as efficient, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.     

Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina. Characteristics of the phosphorylated proteins and their dephosphorylation

To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, we studied cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein. cGMP or cAMP with [gamma-32P]ATP in the dark enhanced the phosphorylation of two ROS proteins with Mr = 10,500 (Band 1) and 8,500 (Band 2) according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg2+. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. The stoichiometry of the phosphate incorporated into Bands 1 and 2 could not be calculated because the amount of Bands 1 and 2 was too small to measure. Both 32P-phosphorylated Bands 1 and 2 (32P-Bands 1 and 2) were solubilized during preparation and the molecular weight of each, in the native preparation, was 19,000. Their isoelectric point was 5.2. The sites of phosphorylation were the serine residue(s). DEAE-Sephadex A-50 column chromatography gave a good separation of Bands 1 and 2 from other 32P-phosphoproteins at 60 mM NaCl. Dephosphorylation of 32P-Bands 1 and 2 in dark-adapted ROS suspension required Mn2+ or Mg2+; the former was more effective than the latter at concentrations below 0.5 mM. Both phosphorylation and dephosphorylation were inhibited by Zn2+.     

Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii.

Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [gamma-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [gamma-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ of Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol in 2 mM. Both ADP and cAMP inhibit the reaction.     

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.     

The effect of cyclic AMP on the growth and morphology of a normal human fibroblast parent strain and its transformed progeny line.

A normal human diploid fibroblast cell strain, Lederle 130 (Led 130), and its virus-transformed progeny line, transformed Led 130, were subjected to 0.75 and 1.5 mM concentrations of adenosine-5'-monophosphate (AMP), cyclic AMP (cAMP) and dibutyryl cyclic AMP (Bt2cAMP). While cAMP was markedly inhibitory to neoplastic cells at 1.5 mM, Bt2-cAMP was even more effective at this concentration, producing 85% inhibition by 4 days and 91% inhibition by 6 days. Bt2-cAMP was the only nucleotide to reverse morphological transformation effects in the neoplastic fibroblasts. Normal fibroblasts were inhibited in growth rate to a comparable extent by all nucleotides, and were not altered morphologically.     

Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii

A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue.

The intracellular level of cGMP was independent of the rate of cell division in cells derived from virally infected brain tissue. The phosphodiesterase inhibitor R07-2956 (4-dimethoxybenzyl-2-imidazolidinone) increased the intracellular level of cGMP in virally infected brain cells, but it did not effect the level of cAMP. There was no correction between the increase in cGMP levels following addition of R07-2956 and changes in mitotic activity in the brain cell cultures. Experimental manipulations which increased the cAMP level were accompanied by a decreased mitotic rate indicating there was a correlation between mitotic activity and the level of cAMP in the same cells. Raising the intracellular level of cAMP by exogenous db-cAMP or cAMP or the use of other phosphodiesterase inhibitors routinely increased the level of cGMP as well. Conversely increasing the intracellular cGMP level by adding the exogenous cGMP increased the level of both cGMP and cAMP.A tissue culture system was used with the cell line derived from viral infected human brain tissue originally obtained from a patient with subacute sclerosing panencephalitis (SSPE). The intracellular levels of cAMP and cGMP were monitored by radioimmunoassay following manipulation of the system by addition of exogenous cGMP (0.05 mM), addition of exogenous db-cAMP (0.5 mM), or cAMP (0.5 mM) and the use of phosphodiesterase inhibitors: theophylline (1.0 mM), papaverine (50 μg/ml), 4-3-butoxy-4-methoxy benzyl-2-imidozalidinone (R020-1724) and R07-2956. Cell division was monitored in treated and non-treated cultures at 24 h intervals by analyzing the cell number and mitotic index.High levels of cGMP were found in cells which were not actively dividing but high levels were just as apt to be present in dividing cells. There was an inverse relationship between cell division and the level of cAMP.     

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys.

The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: properties of the enzyme in crude homogenates

Homogenates of rat neostriatum hydrolysed cGMP faster than cAMP at both high (100 microM) and low (1 microM) substrate concentrations, although the hydrolysis of both nucleotides exhibited similar kinetic properties. Kinetic analysis of the effect of substrate concentration on the rate of cAMP and cGMP hydrolysis gave results characteristic of a negatively cooperative enzyme species, with two apparent Km's for each nucleotide. The ratio between the Vmax of the high Km form and the Vmax of the low Km form was similar in various subcellular fractions of neostriatal tissue, in a preparation of synaptic membranes from whole brain, and in homogenates of other brain regions, including both neural-rich and glial-rich tissues. In homogenates of neostriatum cAMP could almost completely block cGMP hydrolysis and vice versa. The kinetics of this inhibition were competitive at low (1 microM) substrate concentrations, and non-competitive at high (100 microM) substrate concentrations. Various phosphodiesterase inhibitors failed to preferentially inhibit the hydrolysis of either nucleotide at high or low nucleotide concentrations. Preliminary studies of the effect of a Ca(2+)-dependent endogenous activator preparation on the hydrolysis of cyclic nucleotides in homogenates of rat neostriatum showed a specific activation of cGMP hydrolysis at low nucleotide concentrations. The rate of cGMP hydrolysis at 1 microM substrate concentration was doubled in the presence of the activator preparation and 100 microM-CaCl2, while cGMP hydrolysis at 100 microM or cAMP hydrolysis at both 1 microM and 100 microM remained unaffected. These observations raise the possibility that cAMP and cGMP may be hydrolysed by the same enzyme in rat neostriatum, and that an endogenous activating factor may determine the relative affinities of the enzyme for the two nucleotides.     

Cell-free stimulation of the insulin-sensitive cAMP phosphodiesterase by the joint actions of ATP and the soluble fraction from insulin-treated rat liver

The insulin-sensitive cAMP phosphodiesterase (PDE) from rat adipocytes was stimulated 60-70% upon incubation with 2 mM ATP and the soluble fraction (Fraction S-1) from insulin-treated rat liver. The effect of ATP was partially mimicked by ATP-gamma-S or GTP, but not by AMP-PNP. The PDE-stimulating activity in Fraction S-1 was preserved in the presence of 50 mM sodium phenyl phosphate, 50 mM sodium fluoride, and 0.1 mM sodium vanadate. The PDE-stimulating activity was not inhibited with either 0.5 mM H-7 or 5 microM PKI-(5-24)-peptide, but was blocked with 1 mM Kemptide. The active component in Fraction S-1 may be a phosphorylated compound, which, in the presence of ATP, may mediate the hormonal action on PDE.     

Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides

Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment.     

Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

Effect of calcium on the cyclic GMP elevation induced by thymosin fraction 5.

Thymosin fraction 5 induces an increase in cyclic GMP but not cAMP in murine thymocytes. Calcium (0.6 mM) is necessary for an optimal response in both phosphate buffered saline and hepes-buffered RPMI 1640 media. The calcium dependence of the cGMP response was most pronounced in a minimal salts medium (PBS) and higher concentrations (greater than 0.8 mM) caused a lessening of the cGMP elevation induced by thymosin. Basal cGMP levels of thymus and spleen lymphocytes vary with increasing concentrations of calcium (0–1 mM) and to a lesser extent, the levels of cAMP also are increased. Calcium uptake was measured both at mitogenic levels of Con A and at thymosin concentrations which were similar to those necessary for the increase in cGMP. The results suggest that calcium and cGMP play an important role in T cell differentiation under the influence of thymosin.