Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Synthesis of carnitine from ε-N-trimethyllysine in post mitochondrial fractions of Neurospora crassa

An enzyme system in the post mitochondrial fraction of $\text{Neurospora}$$\text{crassa}$ when supplemented with appropriate cofactors formed carnitine from ε-N-trimethyllysine. These findings together with previous studies of ε-N-lysine methylation in this fungi, illustrate that carnitine synthesis in $\text{Neurospora}$ differs markedly in certain features from mammalian systems in that the entire synthesis is carried out employing free intermediates and cytosolic enzymes.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Transport of arginine by an in vitro system

Cell surface glycoproteins of $\text{Neurospora crassa}$ conidia have been shown to bind amino acids and to be genetically associated with the previously defined amino acid transport systems of that organism. L-arginine does not readily permeate a film of $\text{Neurospora}$ conidial lipids. Addition of glycoprotein extracts from $\text{Neurospora}$ to the lipid film enhances permeation of arginine at an initial rate 1000 times the rate of permeation through lipid alone. The initial rate of passage exceeds the rate of unhindered passage (no lipid film) through the same cross sectional area by 10 fold.     

Photoreceptor pigment for blue light responses in Neurospora crassa

Irradiating the mycelium of $\text{Neurospora crassa}$ with blue light causes the photoreduction of a $\text{b}$-type cytochrome. The action spectrum for the photoreduction of the cytochrome $\text{b}$ is similar to action spectra for the photoactivation of carotene synthesis and photoinhibition of the circadian rhythm of conidiation in $\text{Neurospora}$.     

Proteolytic inactivation of a pentafunctional enzyme conjugate: coordinate protection by the first substrate.

The $\text{arom}$ pentafunctional enzyme conjugate of $\text{Neurospora crassa}$ was exposed to trypsin, chymotrypsin, or a protease preparation from $\text{Neurospora}$ in the presence and absence of the first substrate, 3-deoxy-D-$\text{arabino}$-heptulosonate 7-phosphate. It was found that the first substrate coordinately protects all five activities from proteolytic inactivation, which indicates a conformational change induced by this compound. In addition, the data presented are consistent with the “domain” theory of conjugate structure. It is also argued that coordinate protection may be of physiological significance.     

An unusual cyclic AMP-dependent protein kinase from nematodes

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode $\text{Aphelenchus}$$\text{avenae}$. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the $\text{cAMP}\text{cGMP}$ kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.     

Interaction of wild-type and poky mitochondrial DNA in heterokaryons of Neurospora.

The mitochondrial phenotype of $\text{poky}$ and other extra-nuclear $\text{Neurospora}$ mutants is known to predominate over that of wild type in heteroplasms. This phenomenon was investigated in heterokaryons using normally occurring strain differences in restriction enzyme patterns to distinguish wild-type and $\text{poky}$ mitochondrial DNAs. Each of ten independent heterokaryons eventually showed the $\text{poky}$ phenotype as judged by slow growth rate and deficiency of 19 S RNA. Six heterokaryons contained mitochondrial DNAs with restriction enzyme patterns of the $\text{poky}$ parent whereas four contained DNAs which lacked restriction enzyme fragments characteristic of the $\text{poky}$ parent. The latter may be recombinants of wild-type and $\text{poky}$ mitochondrial DNA.     

On the mechanism of tolerance to isoproterenol-induced accumulation of cAMP in rat pineal in vivo.

Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, $\text{in}$$\text{vivo}$, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal $\text{in}$$\text{vivo}$ may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.     

Reciprocal changes in Ca2+/protein activator-dependent and-independent cyclic AMP phosphodiesterase during the development of chick embryos.

The changes in hepatic $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent and independent cAMP phosphodiesterase activity during the development of chick embryo were investigated. Nearly all of the cAMP phosphodiesterase found in the liver during the early embryonic stage was the $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent enzyme; however, this enzyme progressively diminished and the independent enzyme increased proportionately with embryonic development. In the adult liver, little or no $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent enzyme activity could be observed. These results indicate that the concentration of intracellular cAMP may be regulated by these two enzymes in compliance with the development of the chick.     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

Adenylate cyclase and cyclic AMP through the cell cycle of Tetrahymena.

Adenylate cyclase activity and 3′, 5′ cyclic adenosinemonophosphate (cAMP) have been followed through the heat-synchronized cell cycle of Tetrahymena pyriformis. While the specific activity of adenylate cyclase remained essentially constant throughout the cycle, cAMP oscillated (between 10 and 50 pmoles/mg protein) through two cycles. Minima were observed at each division ($\text{D}\text{S}$ border) and maxima at each $\text{S}\text{G}\text{2}$ border. Each heat shock caused slight temporary reduction in cyclase activity. Further observations suggest to us that adenylate cyclase shows conformational changes in response to temperature-induced alterations and to changes in lipid composition of membranes.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

Effect of thyrotropin releasing hormone on the camp content in circumesophageal ganglia of lymnaea emarginata

The effect and metabolic fate of thyrotropin releasing hormone on the cAMP content of $\text{in}$$\text{vitro}$ incubated ganglia from the pond snail $\text{Lymnaea}$$\text{emarginata}$ was studied. It was found that TRH caused an increase in the cAMP content of parietal ganglia, a decrease in the cAMP content of cerebral ganglia and no change in the other circumesophageal ganglia. $\text{In}$$\text{vitro}$ incubated ganglia did not accumulate or degrade significant amounts of ³H-TRH.     

Structure of a ribosomal 5S RNA from a mushroom, Coprinus cinereus

The nucleotide sequence of the 5S rRNA from a mushroom, $\text{Coprinus cinereus}$, was determined to be: pAUCCACGGCCAUACGACUCUGAAAGCACCGCACCGCAUCCCGUCCGAUCUGCGCAGUUAACCAGAGUGCCGCUCAGUUAGUACCACGGUGGGGGACCACGCGGGAAUCCUGGGUGCUGUGGUU. This sequence is consistent with current models for the secondary structure of 5S RNAs and indicates a very high degree of sequence conservation among the most highly evolved fungi. Sequence heterogeneity was not evident in this fungus suggesting that the more highly evolved fungi may not contain the dispersed pattern of 5S rRNA genes which have been observed in intermediate fungi such as $\text{Neurospora}$ (Selker, E.U., and Yanofsky, C. (1981) Cell $\text{24}$, 819–828.)     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) ($\text{5Z}\text{)-9-}\text{deoxy}\text{-6,9α-}\text{epoxy}\text{-Δ}{}^5\text{-}\text{PGF}{}_{\mathrm{1\alpha }}$ has been found to be a potent stimulator of cAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of cAMP accumulation in platelets than PGE₁. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH₂, and a PGH₂-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH₂.     

Cycloheximide inhibition of insulin control of liver glycogen synthase b into a conversion.

Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase $\text{b}\text{into}\text{a}$ conversion without affecting the level of synthase $\text{b}$. The effect of cycloheximide, believed to elevate cAMP in liver of normal rats, is independent of cAMP levels of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase $\text{b}$ to $\text{a}$ conversion by cycloheximide does not appear to be the result of a cycloheximide-induced cAMP-dependent phosphorylation of synthase $\text{a}$ to $\text{b}$ and suggests that insulin control of synthase $\text{b}$ and $\text{a}$ interconversions is dependent upon cycloheximide-sensitive protein synthesis.     

Mechanism of glycogenolytic action of cycloheximide in rat liver

Cycloheximide (0.1 to 0.2 m$\text{M}$) increases cAMP concentration 3 to 4-fold in isolated rat liver slices $\text{in vitro}$. This increase in cAMP concentration parallels an increase in phosphorylase activity. When cycloheximide, at a concentration used to inhibit protein synthesis (1 to 2 μg/g body weight), is administered to whole animals, phosphorylase is activated up to 13-fold by 6 hours. This leads to almost complete depletion of liver glycogen (from about 40 mg/g liver to 0.4 mg/g liver).     

Effect of cycloheximide on adenosine 3':5'-monophosphate level in rat epididymal fat tissue.

Cycloheximide at 0.1 to 0.2 m$\text{M}$ increases cAMP concentration up to five-fold in epididymal fat tissue $\text{in vitro}$. This increase in cAMP concentration is accompanied by a 40% activation of glycogen phosphorylase. Propranolol, a specific β-adrenergic antagonist, blocks the cycloheximide-mediated cAMP increase. Epinephrine stimulates cAMP formation up to 25-fold under the same condition. This increase is also blocked by propranolol. Cycloheximide also partially blocks the epinephrine stimulated cAMP increase, suggesting that both compounds act at the same site.