A simplified assay for cyclic AMP using protein kinase binding

Summary The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.Acknowledgment. This work was partially supported by grants HL-16583 and HL-18827 from the National Heart and Lung Institute.     

Microinjection of synthetic protein kinase inhibitor into single barnacle muscle fibers before and after cyclic AMP

Single muscle fibers from the barnacle Balanus nubilus have been used as a preparation to see if a synthetic 20-residue PKI (5-24)-peptide is able to block or reverse the stimulatory response of the ouabain-insensitive Na efflux to injected cAMP. The results obtained show that this peptide behaves as a powerful inhibitor of the cAMP-mediated response and is also able to partially reverse the sustained stimulation of the Na efflux observed in ouabain-poisoned fibers following the injection of subunit A of cholera toxin.     

Microinjection of synthetic protein kinase inhibitor into single barnacle muscle fibers before and after cyclic AMP

Summary Single muscle fibers from the barnacleBalanus nubilus have been used as a preparation to see if a synthetic 20-residue PKI (5-24)-peptide is able to block or reverse the stimulatory response of the ouabain-insensitive Na efflux to injected cAMP. The results obtained show that this peptide behaves as a powerful inhibitor of the cAMP-mediated response and is also able to partially reverse the sustained stimulation of the Na efflux observed in ouabain-poisoned fibers following the injection of subunit A of cholera toxin.Warmest thanks to Prof. E. H. Fischer and Kurt Diltz for a sample of sPK1. This work received support from NSF, the Graduate School Research Committee and from NIH through a General Research Support Grant to the University of Wisconsin Medical School.     

Hyperpolarization by cyclic AMP (activation of diphosphoinositide kinase)

Zusammenfassung In-vitro-und In-vivo-Versuche beweisen, dass ein molekulärer Mechanismus für das Zustandekommen der Membran-Hyperpolarisation verantwortlich ist.

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Presented in part at the Annual Meeting of the Biophysical Society, Toronto, Canada, February 1972.     

A sensitive protein assay method using micro-titer plates

Summary A commercially available protein-assay, based on the reaction between bicinchoninic acid (4,4-dicarboxy-2,2-biquinoline) and copper was adapted for use in microtiter plates. The assay is sensitive between 10 m/ml and several mg/ml, and it uses only 10 l of sample material. The results obtained with the method were in accordance with other protein determination methods. The assay was shown to be reproducible, reliable and insensitive to non-ionic detergents.Acknowledgments. The authors are grateful to Miss Barbara Moser who skillfully performed a number of these experiments. This research was supported by the Swiss National Science Foundation, Grant No 3.300–0.82, to U.B.     

Allergic reactions, cyclic AMP and histamine release.

Both isobutylmethylxanthine and theophylline increased the level cyclic AMP in the mast cell. Theophylline reduced the allergic histamine release, whereas isobutylmethylxynthine caused a pronounced potentiation of the histamine release. This indicates that the hypothesis of an inverse relationship between the level of cyclic AMP in mast cells and histamine release is too simple.     

Conversion of mammalian cyclic GMP-dependent protein kinase into modulator-dependent protein kinase (type II) in vitro

The spontaneous conversion of mammalian cyclic GMP-dependent protein kinase (G-PK) into modulator-dependent protein kinase (type II) (M-PKII) in the absence of cGMP or histone was observed in vitro. The findings, together with similarity in substrate protein specificity, suggest that M-PKII is the catalytic subunit of mammalian G-PK.     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Intestinal vasoactive peptide receptors in enterocytes : specific binding and stimulation of cyclic AMP]

Receptors for the vasoactive intestinal peptide (VIP) were characterized on enterocytes isolated from Rat small intestine. Native VIP inhibited competitively the binding of 125I-VIP to enterocytes and strongly stimulated cyclic AMP production; both these effects were observed for concentrations of VIP as low as 0.5-1.0 ng/ml (0.15-0.30 nM) which are compatible with the VIP concentration in the gut.     

Flavonoid compounds are potent inhibitors of cyclic AMP phosphodiesterase.

The inhibitory activity of 19 flavonoid molecules on cyclic AMP breakdown by a commercial beef heart phosphodiesterase preparation is reported. 7 compounds are active in the micromolar range, 2 of which have a potency equivalent to that of papaverine. Some structure activity relationships are drawn.     

A rapid assay for the localization of protein kinase modulator (inhibitor) in complete polyacrylamide gels

Summary A gel assay system for determination of inhibitory proteins for protein kinase(s) on isoelectric focusing gels has been developed. Preparations of heat-stable inhibitors were applied, focused, and the complete gels incubated with protein kinase in the presence of substrate protein. At the position where inhibitory protein had focused, the phosphorylation reaction was blocked selectively.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Stimulation of sodium current by cyclic AMP is mediated through protein phosphorylation in Euhadra neurons

The protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and isoquinolinesulfonamide, abolished the inward Na current which was elicited by cAMP.     

Prostaglandin-like substances inPropionibacterium acnes II. Stimulatory effect on ovarian cyclic AMP

Summary The prostaglandin-like substances (PLS) fromPropionibacterium acnes increased the ovarian tissue levels of cyclic AMP (cAMP) approximately 2-fold. The lipid material extracted fromP. acnes thus behaved like PG's of the E-type, and since it is unlikely that other known stimulators of the ovarian cAMP system can be present in the bacterial lipid fraction, these experiments give further evidence in favour of the occurrence of PLS inP. acnes.