Cyclic 3',5'-AMP phosphodiesterase of Neurospora crassa

Cyclic 3′,5′-AMP (cAMP) phosphodiesterase activity can be demonstrated in extracts of $\text{Neurospora}\text{crassa}$. The activity is particulate, has a pH optimum of 7.4, and consists of two forms that have different cAMP binding constants. Methylxanthines, inorganic phosphate, and EDTA are inhibitors of the diesterase as are ATP, ADP, and 8-bromo-cAMP. The enzymatic activity is stimulated by histamine and imidazole. These properties suggest that the $\text{Neurospora}$ enzyme is more closely related to the mammalian than to bacterial cAMP phosphodiesterases.     

Effect of gonadotrophins and testosterone on the seminiferous tubules of the immature rat.

The actions of HCG and PMSG for different periods and of testosterone of the immature rat testis were studied. Short-term administration of HCG (1-3 days) induced an early meiotic and postmeiotic stimulatory effect but a decrease in spermatogonial numbers. Administration of HCG for longer periods (10 days) caused a reduction in numbers of all cell types. Treatment with HCG + PMSG reduced the amount of inhibition, while PMSG alone resulted in histological and humoral signs of stimulation of the interstitial tissue and the meiotic and postmeiotic stages; the numbers of spermatogonia were not affected. Testosterone caused stimulation of the meiotic and postmeiotic stages and a reduced number of spermatogonia. It is concluded that while PMSG directly stimulates spermatogonia, HCG acts through testosterone secretion at the meiotic and postmeiotic stages. The early inhibitory effects of HCG and testosterone on spermatogonial numbers could be ascribed to the inhibition of endogenous FSH by androgens.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Separation of cyclic 3',5'-AMP from ATP, ADP, and 5'-AMP by precipitation with inorganic compounds

The antibiotic anisomycin is a very useful tool in studying protein synthesis since it is a specific inhibitor of the peptidyl transferase centre of eukaryotic ribosomes (5–7). By tritium exchange labeling followed by chromatographic and electrophoretic purification, we have obtained [³H]anisomycin of specific activity 285 mCi/mmole, and the methodology followed is described in this paper. This method is useful in preparing tritium labeled antibiotics other than anisomycin provided that the nonradioactive compound has the following characteristics: (a) a chemical structure resistant to the method required for tritium labeling, (b) ionic groups, and (c) chromophore groups with absorption maxima in the uv or visible part of the spectrum. Since these circumstances concur frequently in a number of chemical structures, a method essentially similar to that described in this work might be widely used. The method was not applicable to amicetin, blasticidin S, and fusidic acid, as these antibiotics were broken down during the tritium labeling. However, gougerotin, a well known inhibitor of peptide bond formation by prokaryotic and eukaryotic ribosomes (2–7), has been tritiated and purified following a method very similar to that described in this contribution to [³H]gougerotin (110 mCi/mmole) (16).     

The effect of diisopropylfluorphosphate on the adenylate cyclase activity and the 3',5'-AMP content of myocardium and brain]

Adenylate cyclase [ATP-pyrophosphatelyase (cyclizing) EC 4.6.1.1.] from ventricular muscles and the cerebrum of rats can be inhibited by diisopropylphosphofluoridate (DFP) and paraxone in concentrations from 10(-7) - 10(-9)M, and by dimethoate in concentrations from 10(-5) - 10(-7)M. The cAMP content in the heart diminished after i.p. 1.44 mg/kg DFP by 59.9%, and in the cerebrum by 68.2%. The depletion of cAMP caused by double LD50 DFP in the rat heart can be influenced neither by atropine alone, nor in combination with TMB-4. The results are discussed with regard to a possible action mechanism.     

Specific binding of 3',5'-AMP by rat liver mitochondria

It was found that 3':5'-AMP is bound by rat liver mitochondria with an affinity which corresponds to a physiological concentration of the nucleotide and a low capacity. The bound 3':5'-AMP rapidly dissociates upon dilution of mitochondrial suspensions. This finding points to the existence in mitochondria of a 3':5'-AMP receptor protein. The putative biological role of this protein is discussed.