A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

High and low affinity binding of folate to proteins in serum of pregnant women

Binding of [³H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37°C). A Scatchard analysis revealed the presence of high-affinity (K_ass = 10¹⁰M^?1, N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (M_r ≈ 30 000–35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/ 30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (M_r > 200 000) protein, probably α₂-macroglobulin.High-affinity binding which displayed positive cooperatively was saturated at folate concentrations above 10^?10 M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N¹⁰-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10^?10–10^?7 M. Dissociation of folate was rapid.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Equilibrium constants under physiological conditions for the condensation of acetaldehyde with tetrahydrofolic acid

The nonenzymatic reaction of ethanol-derived CH₃CHO with tissue constituents continues to be of interest as a potential mechanism underlying the toxicity of alcohol. The current study has focused on the spontaneous condensation of CH₃CHO with H₄folate under physiological conditions (38 °C, pH 7.0, I = 0.25 M). Computer analysis of uv spectral changes with increasing CH₃CHO concentrations demonstrated the presence of at least two different adducts. The observed equilibrium constant (K_obs) for the formation of the first adduct is 91 ± 2 m^?1 (121 ± 2 m^?1 at 25 °C), a value which is unaffected by variations in ionic strength (0.06–1.0 m) or by free [Mg²⁺] up to 5 mm. The NMR spectrum is compatible with the structure: 5,10-CH₃CH-H₄folate analogous to the naturally occurring 5,10-CH₂-H₄folate. The formation of the latter compound from HCHO and H₄folate, however, is much more favorable under the same conditions [K_obs = 3.0 ± 0.2 × 10⁴ M^?1 (38 °C), 3.6 ± 0.1 × 10⁴ M^?1 (25 °C)]. At the levels of CH₃CHO which accumulate during ethanol metabolism in vivo only a small fraction of the H₄folate will exist as the CH₃CHO derivative, yet it may ultimately be the ratio of free CH₃CHO to free HCHO in tissue which determines the physiological importance of the CH₃CHO adduct. Other adduct(s) of CH₃CHO with H₄folate are observed at very high levels of CH₃CHO but are unlikely to be of physiological significance.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Purification and some properties of cytosine deaminase from Salmonella typhimurium

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogenous. The molecular weight of cytosine deaminase was determined to be approx. 230 000 containing four identical subunits with each subunit having a molecular weight of 54 000. Cytosine deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50°C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The K_m and V values for cytosine were 0.74 mM and 47.16 μmole/min, respectively. As effectors of enzyme activity, PP_i stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171–1174).     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Purification and characterization of adenosine deaminase from Klebsiella sp. LF 1202

Adenosine deaminase was induced when the cells of Klebsiella sp. LF 1202 were cultured in the medium containing adenosine as a sole source of carbon and nitrogen. The induction was partially repressed by the addition of ammonium sulfate in the medium. The amount of adenosine deaminase reached approximately 4.6% of the total intracellular soluble proteins. The enzyme was purified approximately 22-fold with a 25% activity yield. The enzyme was a monomer with a molecular weight of 26,000. The optimal activity was obtained at pH 8.0, 37°C, and the K_m value for adenosine was 37 μM. Metal ions such as Zn²⁺, Co²⁺, Fe² and Ni⁺ inhibited the activity of the enzyme. Sulfhydryl blocking agents such as p-chloromercuribenzoate and HgCl₂ were also found to be potent inhibitors for adenosine deaminase.     

The formation constants of the proton and calcium complexes of prostaglandins PGE1 and PGF1b

The formation constants of the proton and calcium complexes of PGE₁ and PGF_1β have been measured in aqueous solution at 20 ± 1°. The proton complexes are shown to be effectively ionized at pH 7, log K_HL values being 5.12 and 5.46, respectively. The calcium complexes, studied radiometrically using ⁴⁵Ca by ion exchange equilibria, have formation constants (K_CaPG) of 37 and 9 (approx.), respectively. “Model” ligands studied in developing the technique were 3-hydroxybutyric acid (K_Ca, 3.1) and 4-hydroxybutyric acid (K_Ca, 5.7).     

Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Binding of folates to Dictyostelium discoideum cells. Demonstration of five classes of binding sites and their interconversion

Studies of the binding of four folate derivatives to the cell surface of Dictyostelium discoideum indicate the existence of five types of sites. About 99% of the total number of binding sites (160 000 per cell) belongs to the ‘non-selective’ type, which recognizes folate, 2-deaminofolate and methotrexate with equal affinity. As judged by the kinetics of association and dissociation this class consists of two distinct subtypes; a high-affinity site, designated by A^H, and a low-affinity site A^L. Upon addition of ligand a number of the low-affinity sites is converted to the high-affinity state. Prolonged dissociation revealed the presence of extremely slowly dissociating sites. While the A-sites released bound ligand within 5 s, the slow (B) type yielded a half-time of about 6 min. This class (550 sites per cell) showed a clear selectivity for the four folates, with N₁₀-methylfolate being the best ligand. From the kinetics of association and dissociation it is concluded that the B-sites are interconvertible with another binding type. In addition a class of sites was detected, which binds N₁₀-methylfolate and folate with high affinity but 2-deaminofolate and methotrexate with approx. 100-fold lower affinity. Kinetic studies reveal that this C-class is also composed of two subtypes; a fast equilibrating site (within 1 s) designated as C^F, and a slower site C^S. It is proposed that before binding of ligand only C^F exists, while after binding this binding type is converted into C^S. At equilibrium more than 90% of the C-sites have attained the C^S state.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

Bicarbonate effect on electron flow in a cyanobacteriumSynechocystis PCC 6803

In this communication, evidence is presented from the kinetics of Q_A ^? decay (where Q_A is the first plastoquinone electron acceptor of photosystem II) and oxygen evolution for the requirement of bicarbonate in the electron transport in a cyanobacteriumSynechocystis (Pasteur Culture Collection 6803). A large slowing down of Q_A ^? oxidation, measured from the variable chlorophylla fluorescence after saturating actinic flashes, was observed in the thylakoids ofSynechocystis 6803 depleted of bicarbonate in the presence of 25 mM formate. Qualitatively similar results were obtained with DCMU-treated thylakoids. This shows that bicarbonate depletion inhibits electron transport on the acceptor side of photosystem II between Q_A and the plastoquinone (PQ) pool in cyanobacteria. Addition of 2.5 mM HCO₃ ^? fully reversed the inhibition of electron flow caused by bicarbonate depletion. Two exponential phases of Q_A ^? decay, a fast one and a slow one, were observed with halftimes of approx. 400 μs (fast) and 26 ms (slow) at pH 6.5. At pH 7.5, these phases were approx. 330 μs (fast) and 21 ms (slow), respectively. The amplitude, but not the halftime, of the fast component decreased by about 70% (pH 6.5) or 50% (pH 7.5); this was accompanied by a concomittant increase in the slow phase. Twenty mM bicarbonate stimulated, by a factor of 4, the Hill reaction in bicarbonate-depletedSynechocystis cells. This effect is independent of CO₂ fixation as it was observed even in the presence of an inhibitor DBMIB.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP