Biotin analogs activate guanylate cyclase

Summary The sulfur atom in the vitamin biotin has previously been suggested to be essential in biotin's mechanism of action. In a series of investigations on structure-function relationships with biotin analogs not containing the sulfur atom, the biotin analogs, azabiotin, bisnorazabiotin, carbobiotin and isoazabiotin enhanced guanylate cyclase, an enzyme that has recently been demonstrated to be activated by biotin. These analogs increased guanylate cyclase activity two-fold in liver, cerebellum, heart, kidney and colon at 1 M concentrations. The ED₅₀ for stimulation of guanulate cyclase activity occurred at 0.1 M for each of the biotin analogs. These data indicate that the sulfur atom is not essential in biotin's activation of guanylate cyclase since these analogs do not contain the sulfur atom. Studies on the ring structure of biotin revealed that even compounds with a single 5-membered ring (2-imidazolidone) could augment guanylate cyclase activity. The guanylate cyclase co-factor manganese was not essential for the enhancement of guanylate cyclase by these agents but a maximal activation of this enzyme by these analogs could not be obtained without manganese present.     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

Derivatives of Benzotetrazine-1,3-dioxide Are New NO-donors, Activators of Soluble Guanylate Cyclase, and Inhibitors of Platelet Aggregation

The ability of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides to generate nitric oxide (NO) and activate soluble guanylate cyclase was investigated. All of these compounds were found to be thiol dependent NO-donors and guanylate cyclase activators. The maximal stimulatory effect of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides was observed at 10 M concentration and the activity increase was 4.5-, 15.0-, and 8.2-fold in the presence of 20 M dithiothreitol and 11.3-, 31.6-, and 20.5-fold, respectively, in the presence of added glutathione (100 M). The NO-dependent mechanism of benzotetrazine-1,3-dioxide nitroderivative-induced activation of soluble guanylate cyclase (in the presence of 100 M glutathione) was confirmed by the inhibition (by 78%) of 7-nitrobenzotetrazine-1,3-dioxide (10 M)-stimulated guanylate cyclase activity in the presence of the NO-scavenger-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO, 50 M) and by the inhibition with 1H-[1,2,4 ]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.3 M) of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides (10 M)-stimulated guanylate cyclase by 34, 69, and 39%, respectively. All compounds used inhibited ADP-induced aggregation of human platelets with IC ₅₀ of 10.0, 1.3, and 2.0 M for 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides, respectively. A clearly defined correlation was established between the ability of the compounds to generate NO, activate soluble guanylate cyclase, and inhibit platelet aggregation.     

A new type of formaldehyde-induced fluorescence in a population of endocrine cells in cat antro-pyloric mucosa

Summary Formaldehyde treatment followed by acidification (formaldehyde-HCl) revealed an extensive system of intensely fluorescent endocrine-like cells in the mucosa of the cat pyloric gland area. Maximum excitation was at 440–450 m and maximum emission at 500–520 m. The cells were barely visible upon treatment with formaldehyde alone or with formaldehyde-ozone. Hence, the properties of the fluorophore in the cat antrum differ from those of all hitherto reported formaldehyde-induced fluorophores.The cells described were argyrophil (but non-argentaffin), they stained with lead-hematoxylin, and they showed green formaldehyde-induced fluorescence after injecting the animal with l-DOPA. It is possible that the formaldehyde-HCl fluorescent cells are identical with the cells believed to store gastrin.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

Kinetics of the transhydrogenase reaction catalyzed by mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The kinetics of the NADH3'-acetylpyridine adenine dinucleotide (APAD⁺) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADHAPAD⁺ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD⁺ (K _m = 7 M, for SMP) and low rate (V _m = 0.2 mol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V _m = 1.7 mol/min per mg protein and K _m = 160 M, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD⁺. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.     

Elektronenmikroskopische Untersuchung über die Differenzierung der Interzellularsubstanz der menschlichen Lederhaut

Zusammenfassung Es wurde Haut aus der Bauchdecke des Menschen vom 8 cm-Keimling (Scheitel-Steiß) bis zum 82jährigen elektronenmikroskopisch untersucht.Die Differenzierung der Haut wird mit Hilfe der Kriterien Fibrillendicke, Versilberungsmodus und Verhalten der Kittsubstanz verfolgt. Die Differenzierung der Kollagenfibrillen der Haut ist bereits intrauterin abgeschlossen und entwickelt sich nur noch wenig im frühesten Kindesalter (Neugeborenes) weiter.Im Verlaufe dieser Entwicklung werden die Fibrillen dicker, die Kittsubstanz nimmt ab. Bei einem Foeten von 33,4 cm Gesamtlänge hegt der Versilberungsmodus der reifen kollagenen Fibrillen, nämlich die streng periodische Einlagerung der Silberteilchen in die D-Teile.Im hohen Alter werden die Fibrillen dünner, die periodische Innenversilberung wird ungleichmäßig und die Menge der amorphen Kittsubstanz nimmt wieder zu, wobei diese grobschollig ist. Dieser Befund wird diskutiert.Aus der Verteilungskurve der Fibrillendicken geht hervor: Die Fibrillendicken vom 8 cm-Keimling bis zum Neugeborenen schwanken zwischen 5 und 70 m. Im Laufe dieser kontinuierlichen Dickenzunahme wandert das Maximum von 10 und 20 m (Keimling 8 cm Scheitel-Steiß) bis zu 50 m (Neugeborenes). Im Erwachsenenalter schwanken die Fibrillendicken von 30–100 m mit einem Maximum bei 60 m. Im hohen Alter (72–82 Jahre) liegen die Dickenwerte zwischen 20 und 80 m mit dem Maximum zwischen 50 und 60 m.Die Querstreifungsperiode betrug im Durchschnitt 65 m.Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft. Dissertation unter Leitung von Prof. Dr. W. Schwarz.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Whole-body retention,and urinary and fecal excretion of mercury after subchronic oral exposure to mercuric chloride in rats

The effects of long-term daily intake of mercury on its urinary and fecal excretion, whole-body retention, and blood concentration in male rats were observed. The animals were exposed to mercuric chloride labeled with ²⁰³Hg via drinking water for 8 weeks (5, 50 and 500 m Hg). ²⁰³Hg in urine, feces and blood was quantified. The blood mercury concentration did not keep a linear relationship with the increasing dose. The percentage of the total amount of mercury intake which is excreted by the fecal route in rats exposed to 500 m Hg was significantly lower than in those exposed to 5 and 50 m. The daily dose percentage of mercury excreted in urine increased with dose size. The results show that the absorption fraction of mercury through the gastrointestinal tract (30–40%) was higher than values previously reported.     

Properties of cellulases fromSclerotium rolfsii

Summary Filter paper cellulase (FPCase) and carboxymethyl cellulase (CMCase) activities ofSclerotium rolfsii were maximal at pH 4.8 and 3.6, respectively, and at 55°C. Both activities increased three-fold when the concentrations of filter paper and carboxymethylcellulose were increased by six-and five-fold, respectively. Enzyme activities decreased with increasing incubation period though sugar formation increased. The K_m and V_max values activity were 61.5 mg and 0.57 m for FPCase and 6.25 mg and 1.58 m for CMCase.

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Propriétés des celluloses de Sclerotium rolfsii

Résumé Les activités cellulolytiques mesurées sur papier filtre (FPCase) et avec la carboxyméthylcellulose (CMCase) chezSclerotium rolfsii ont leur valeur maximum respectivement à pH 4.8 et 3.6, et à 55°C. Les deux activités augmentent de trois fois quand on augmente les concentrations en papier filtre et en carboxymethylcellulose de six et cinq fois respectivement. Les activités enzymatiques décroissent lorsque la période d'incubation augmente, bien que la formation de sucre croisse. Les valeurs de K_m et V_max sont de 61.5 mg et 0.57 m pour la FPCase et de 6.25 mg et 1.58 m pour la CMCase.

     

Reversible activation of the ATP-dependent potassium current with dialysis of frog atrial cells by micromolar concentrations of GDP

Summary We studied the effects of internal and external solutions on potassium currents in frog atrial cells. Experiments were carried out in whole cell recording in the presence of tetrodotoxin and cobalt in the bath to suppress the inward currents. In the absence of pyruvate and glucose in the external solution, a time-independent current increased progressively in a few minutes till the death of the cell. This current had the properties of the ATP-sensitive potassium current IK(ATP) in mammalian cells. In the presence of pyruvate and glucose in the external solution, the membrane current stayed low for 30 min. Addition of guanosine monophosphate (GMP, 40 m), guanosine triphosphate (GTP, 40 to 1000 m), adenosine diphosphate (ADP, 40 m) or adenosine triphosphate (ATP, 3000 m) to the internal solution had no major effect on the current amplitude. In contrast, addition of GDP (20 or 40 m) produced a loss of rectification in a few minutes. The current activated by GDP was time independent as was the current observed in the absence of glucose and pyruvate. It was sensitive to cesium and barium, it was blocked when ATP was added to GDP in the internal solution, and it was suppressed by the sulphonylurea glibenclamide (1 m). We suggest that GDP produced a local depletion of ATP, by displacement of the equilibrium between ATP, GDP, ADP and GTP. This hypothesis is supported by the fact that the current activated by GDP was rapidly suppressed when adding GTP in excess to the internal solution.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Interaction of phlorizin and sodium with the renal brush-border membraned-glucose transporter: Stoichiometry and order of binding

Summary The order and stoichiometry of the binding of phlorizin and sodium to the renal brush-border membraned-glucose transporter are studied. The experimental results are consistent with a random-binding sites is one-to-one. When the kinetics of phlorizin binding are measured as a function of increasing sodium concentration no significant variation is found in the apparent number of binding sites; however, the apparent binding constant for phlorizin decreases rapidly from approximately 16 m at [Na]=0 to 0.1 m at [Na]=100mm and approaches 0.05 m as [Na]. The experimental data are fit to a random carrier-type model of the coupled transport of sodium andd-glucose. A complete parameterization of the phlorizin binding properties of this model under sodium equilibrium conditions is given.     

Effect of vanadate on brain protein phosphorylation

The effect of vanadate on the phosphorylation of synaptosomal membrane proteins prepared from rat cerebral cortex was studied. Vanadate concentrations of 10^–6, 10^–5, and 10^–4 M increased the endogenous phosphorylation activity by 25%, 37%, and 75%, respectively. Increasing the ATP concentration in the assay medium from 50 to 500 M did not influence the above effect. A commercial preparation of the purified protein kinase was stimulated 40% by 10^–3 M vanadate. Calcium-calmodulin dependent activity was stimulated only 20% by 10^–5 M vanadate. The effect was not enhanced by further increasing vanadate concentration. Addition of calcium ions (above 50 M) suppressed the vanadate effect, while an inhibition was observed at high Ca²⁺ concentration (2.5 mM). Below 50 M calcium ions stimulated phosphorylation activity in the absence of vanadate and did not affect the stimulatory action of vanadate. Cyclic AMP-dependent endogenous phosphorylation was also stimulated by vanadate. Activation by cAMP could not be observed at vanadate concentrations above 10^–6 M. Possible mechanisms of the vanadate effect are discussed.