Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Preparation of renal cortex basal-lateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-ATPase. However, the specific activity of (Na⁺ + K⁺)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na⁺ + K⁺)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na⁺ + K⁺)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN₃ plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na⁺ + K⁺)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

A new solid-state microelectrode for measuring intra-cellular chloride activities

Solid-state microelectrodes for measuring intracellular Cl^? activity (aⁱCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 μm), coating them under vacuum with a 0.2–0.3 μm thick layer of spectroscopic grade silver, and sealing them (except for the terminal 2–5 μm of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0 ± 0.6 mV (S.E.) per decade change in α_Cl. Tip resistance was (77.1 ± 3.1 × 10⁹ω (n=30). Electrode response was rapid (10–20 s), was unaffected by HCO₃^?, H₂PO₄^2? or protein, and remained essentially unchanged over a 24-h period. αⁱ_Cl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, αⁱ_Cl significantly exceeded the value corresponding to equilibrium distribution of Cl^? across the cell membrane.     

Purification and succinylation of cyclic GMP from large volume samples and radioimmunoassay of succinyl cyclic GMP.

Improved procedures for isolation of cyclic GMP and cyclic AMP and radioimmunoassay of cyclic GMP with succinylation are described. Procedures involved include modified chromatography on alumina and succinylation of cyclic GMP followed by purification of succinyl cyclic GMP on a Dowex AG 1×8 column. These procedures are convenient and applicable to any volume up to 50 ml of tissue extracts and especially for isotonic incubation mixtures. This assay system is sensitive to 6 femtomoles of cyclic GMP/tube. On radioimmunoassay, free and antibody bound [¹²⁵I]-labeled cyclic GMP are separated by Millipore filtration. Cyclic GMP levels in several tissue samples were determined in order to show the applicability of the procedures.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Inhibitory effect of 8-substituted adenosine derivatives on Ca++ and modulator protein-dependent phosphodiesterase activity.

8-Substituted adenosine and cyclic AMP derivatives exhibited some negative Cotton effects in circular Dichroism at B_2u band in pH 7.5 solution, suggesting that these derivatives take syn conformation. The adenosine derivatives, as well as cyclic AMP derivatives, competitively inhibited the cyclic AMP hydrolyzing activity in Ca⁺⁺ and modulator protein-dependent phosphodiesterase preparation from hog brain cortex. The inhibitory potential of an adenosine derivative was lower than that of the cyclic AMP derivative having the same substituent by the lack of the phosphate moiety for which affinity was 0.5 kcal / mol. These results may suggest that the cyclic AMP hydrolyzing site on the enzyme requires the syn conformation of purine riboside.     

Phosphorylation of Escherichia coli RNA polymerase by rabbit skeletal muscle protein kinase

Rabbit skeletal muscle protein kinase catalyzes the phosphorylation of DNA-dependent RNA polymerase of Escherichia coli in the presence of adenosine 3′,5′-monophosphate and ATP. The phosphorylation occurs on one (or more) serine residue(s) in the σ-factor under reaction conditions similar to those employed for RNA synthesis. The phosphorylation of RNA polymerase and its stimulation by protein kinase are inhibited by a specific heat-stable inhibitor from rabbit skeletal muscle. With conditions more favorable for the protein kinase reaction, phosphorylation of RNA polymerase also occurs on the β subunit of the core enzyme, but this reaction occurs at a much slower rate than the phosphorylation of the σ-factor.     

Variations du taux d'amp cyclique et des activités spécifiques de l'adénylate cyclase et de l'amp cyclique-phosphodiestérase pendant le cycle cellulaire d'un actinomycète

The variations in the concentrations of intra- and extracellular cyclic AMP and in the specific activities of adenylate cyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) have been monitored in synchronized culture of Nocardia restricta, a prokaryote belonging to the group of Actinomycetes. At the beginning of the cell cycel, during a first period of RNA and protein synthesis, there is an increasing synthesis of adenylate cyclase which can be suppressed in the presence of chloramphenicol or rifampicin. Simultaneously, the specific activity of cyclic AMP phosphodiesterase decreases and the concentrations of intra- and extracellular cyclic AMP rise. After the end of DNA replication, during a second period of RNA and protein synthesis, the specific activity of cyclic AMP phosphodiesterase increases; during the same time, the specific activity of adenylate cyclase and the level of intracellular cyclic AMP drop. It appears that the overall metabolism of cyclic AMP is coordinated so that the cyclic AMP level will be high at the beginning of DNA replication and will fall thereafter. The results are discussed in comparison with known data about the variations of cyclic AMP during the cell cycle of mammalian cells in cultures.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Guanosine 3′,5′-monophosphate in fruits of Evodia rutaecarpa and E. officinalis

Extraordinary high levels of cGMP activity were detected in the fruits of Evodia rutaecarpa and E. officinalis. The mature fresh fruits contained a cGMP-like substance in concentrations ranging from 10 to 35 mmol/g dry wt, as determined by both a competitive binding assay and radioimmunoassay. The partially purified cGMP-like substance from E. rutaecarpa showed the same chromatographic properties (TLC and columns) as authentic cGMP and was decomposed by cyclic nucleotide-specific phosphodiesterase.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N²-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

In vivo binding of 2,3,6,2′,3′,6′-hexachlorobiphenyl and 2,4,5,2′,4′,5′-hexachlorobiphenyl to mouse liver macromolecules

The role of metabolic activation in the binding of polychlorinated biphenyls (PCBs) to cellular macromolecules was investigated in vivo by comparing the relative binding of 2,4,5,2′,4′,5′-[U-¹⁴C]hexachlorobiphenyl (2,4,5), a slowly metabolized PCB, with that of 2,3,6,2′,3′,6′-[U-¹⁴C]hexachlorobiphenyl (2,3,6), a rapidly metabolized PCB, and the appropriate controls. Each hexachlorobiphenyl was administered to mice, orally for 5 days (7.28 mg/kg/day). Following the dosing schedule, animals were killed at 1, 5 and 8 days. The concentration of each PCB was determined in liver, muscle and kidney and in purified macromolecules isolated from those tissues. The concentration of 2,4,5 was consistently higher than the concentration of 2,3,6 in all tissues studied. However, the amount of 2,3,6 bound to the purified macromolecules was consistently at least one order of magnitude greater than that of 2,4,5. The greatest binding was observed in RNA followed by protein and DNA, respectively. The purity of the macromolecules and the presence of PCB-derived radioactivity at the monomer level were confirmed. This is the first report of ¹⁴C-labeled PCB being bound to purified RNA, DNA, and proteins isolated from the tissues of animals treated in vivo. The binding is thought to be covalent and to be the result of metabolic activation.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.