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An o -diphenol:O 2 oxidoreductase has been purified about 40-fold from potato tuber slices. The enzyme differs from previously described polyphenoloxidases purified from potato for the fact that chlorogenic acid and 4-methylcatechol are much better substrates than DOPA and catechol. The name of chlorogenic acid oxidase is proposed for this enzyme. The K m values for chlorogenic acid and for O 2 are, respectively, 1·7.10 −4 M and 1·5.10 −4 moles/1. The action of inhibitors and of the pH on the activity of the enzyme have been studied.

The reaction of the peptidyl-tRNA in an “in vitro” system from Neurospora crassa with puromycin has been studied by different experimental approaches. Ribosomes precharged with labelled polyphenylalanine have been prepared and the release of radioactivity, occurring after the reaction with puromycin, has been followed on sucrose density gradients. Furthermore the reaction of endogenous peptidyl-tRNA carried by ribosomes isolated from actively growing mycelia with 3H-puromycin has been characterized. By this latter technique it has been possible to evaluate the percentage of ribosomes engaged in protein synthesis in ribosomal populations isolated from mycelia in different stages of growth.

Changes of respiratory metabolism in developing castor bean seeds. — The respiratory metabolism and the effect of the removal of the teguments on gas exchanges in castor bean seeds at various stages of development has been investigated. Maximal values of oxygen uptake rate were found in the period of fat accumulation. From this period on, oxidative activity steadly decreased to become, in the mature seeds, almost undetectable. In all stages of maturation, the removal of the teguments induced a decrease of the respiratory rate of the seeds. This finding indicated that the respiratory rate of the internal tissues is not directly limited by a scarce availability of oxygen caused by a low permeability of the teguments to gases. The value of the respiratory quotient (R.Q.) was found close to 1 in the first stages, during the growing of the endosperm while in the following period of fat accumulation it rised to 1.6, and falled again, just before the abscission of the seed, to 0.7. This fall of the R.Q....

Effects of inhibitors of protein synthesis on the development of metabolic activity in the endosperm during the germination of castor bean seeds. — The effect of chloramphenicol, streptomycin and actinomycin-C on the increase of the activities of glyceroaldehyde-phosphate dehydrogenase, aldolase, glucose-6-phosphate dehydrogenase, fructose 1–6 diphosphate-1-phosphatase, phosphomonoesterase, in the endosperm of germinating castor bean seeds was investigated. In all cases, the protein synthesis inhibitors depressed the activation of the enzymes tested: in particular, actinomycin (50 μg/ml) completely suppressed the increase of the activities. The development of the rate of oxygen uptake and the conversion of fats to sugars was strongly affected by the inhibitors. These data suggest that the increase of the activities of several enzymes in the germinating endosperm is dependent on enzyme synthesis rather than on the conversion from the inactive to the active form of the enzymes.

A method for preparing ribosomes from Neurospora crassa is described in which che ribosomes prepared are free from the enzymes cytochrome c oxidase and NADPH-cytochrome c reductase, DNA and phospholipid material.

The effects of oxygen and of dinitrophenol on the respiratory activation of potato tuber slices. — It is well known that thin slices from storage organs show, in the first period following preparation and washing, a transition from a low to a high level of metabolic activity. In the potato tuber discs (and similar materials) this transition is characterized by: a) a several fold increase of oxygen uptake; b) an increase of the capacity of dissimilating to CO2 the carbon 1 of glucose; c) a decrease of the sensibility of oxygen uptake to the uncoupler 2,4 dinitrophenol (DNP); d) an increase of the capacity of ion and sugar uptake; e) the development of marked capacity of protein synthesis; f) and increase of the activity (as determined in homogenates) of several enzymes involved in the directs oxidation of glucose-6-P or — possibly — in the reoxidation of the TPN reduced by the pentose phosphate pathway reactions. The two latter responses appear with some delay respect the activation of oxygen upta...

The preparation of active subunits of ribosomes isolated from mycelia of Neurospora crassa has been studied. Ribosomes extracted from non-growing mycelia and consisting essentially of monosomes are completely dissociated into 60- and 40-S subunits by treatment with a high concentration of KCl. Ribosomes isolated from growing mycelia consist both of mono- and polysomes and are only partially dissociated polysomes largely unaffected by the treatment with KCl. Pretreatment of the growing ribosomes with puromycin makes them all susceptible to the dissociating action of KCl, suggesting a stabilizing action of the peptidyl-tRNA. The subunits prepared by such a treatment are able to reassociate, yielding ribosomes active in the poly(U)-directed polymerization of phenylalanine. The activity of growing and steady ribosomes has been shown previously to depend in a different way upon the concentration of Mg2+. Ribosomes reconstituted from growing and steady subunits maintain a different activity at low Mg2+ concentration. By reconstitution of hybrid ribosomes it is demonstrated that a higher activity at low Mg2+ concentration is always found when the 60-S subunit is derived from growing ribosomes. Treatment of the ribosomes with puromycin abolishes this effect. It is suggested that the presence of the peptidyl-tRNA bound to the 60-S subunit induces an increased phenylalanine polymerization at low Mg2+ concentration.

Recent experiments comparing the activities of ribosomes isolated from the same tissue or cells in different stages of growth suggest that functional modifications of the ribosomes may occur during development. Ribosomes isolated from Neurospora crassa mycelia having different rates of growth show differences in the amino acid incorporating activity. In the absence of exogenous mRNA the ribosomes isolated from the growing mycelia present a much higher incorporating activity than the steady ones. Moreover the poly U-directed phenylalanine polymerization shows a different Mg++ dependence according to whether the ribosomes are isolated from actively growing or from steady mycelia. The different pattern of the Mg++ dependence is observed also when the ionic composition of the reaction mixture is changed (the buffer Tris is substituted with HEPES, spermidine is added) so that the overall efficiency of the system increases several folds. While the different endogenous activities can be easily explained...

Aumento della sintesi di invertasi in seguito a trattamento con saccarosio in cotiledoni isolati da semi germinanti di ricino. – L'attivita invertasica per cotiledone aumenta durante la germinazione di semi di ricino. In cotiledoni isolati ed incubati in acqua distillata per 15–22 ore, l'aumento di attivita invertasica e molto scarso, L'aggiunta di saccarosio 0,1 M al mezzo di incubazione provoca un aumento di circa 40% dell'attivita invertasica; aumento che non si riscontra se i cotiledoni vengono incubati in glucosio 0,1 M. La pre-senza di attinomicina D e di puromicina nel mezzo di incubazione previene lo sviluppo dell'attivita invertasica. L'apparente specificita del saccarosio nell'indurre l'aumento di sintesi dell'enzima viene brevemente discussa nel quadro piu ampio dei fenomeni di regolazione da substrato delle sintesi di enzimi.

Respiratory activity, enzyme levels and water contents in the ripening castor beans seed endosperm. — In the endosperm of the developing castor bean seed oxygen uptake, water contents and the « in vitro » measurable activity of various enzymes parallely drop during the terminal strages of ripening. The present investigations shows that also the capacity of water uptake decreases (and that of water loss increases) during ripening. When developing seeds at a stage close to ripeness are removed from the fruits and incubated under a condition of easy water availability, both respiration and enzyme activities rapidly rise; while this is not observed for seeds removed from the fruit at an earlier stage of development. These results are interpreted as indicating that the dehydration of the seed during ripening is both a consequence and a cause of the inactivation of enzyme systems.

The i.r. spectra of some 2-thiophene-carboxamides have been investigated for identification of the N-H stretching, CO stretching and N-H deformation bands. The u.v. spectra have been recorded in order to get information about the chromophores responsible for absorption.

INHIBITION OF GLUCOSE-6-P DEYDROGENASE BY OXIDATION PRODUCTS OF NATURAL PHENOLS. — The inhibition of Gl-6-P dehydrogenase activity found in highly concentrated homogenates from potato tuber tissue, seems due to natural occurring orto-di-quinones, such those arising from polyphenoloxidase catalized oxidation of chlorogenic acid, caffeic acid, and tyrosine. These compounds strongly inhibit Gl-6-P dehydrogenase activity of the potato tuber homogenate as well as that of purified Gl-6-P dehydrogenase from yeast. The inhibition effect appears much stronger when the diphenols are oxidized in the presence of the Gl-6-P dehydrogenase. Gl-6-P and TPN efficently protect the enzyme from the inhibition. Ascorbic acid prevents the inhibition by mantaining the diphenols in the reduced form, but is ineffective in reversing the inhibition.

The changes of the total RNA occurring during germination in potato tubers have been investigated. During germination the development of the new sprouts is accompanied by a progressive decrease of RNA. A preferential breakdown of high molecular weight RNA has been observed. Tubers deprived of the buds mantain the level of RNA of the quiescent tuber. Gibberellic acid treatment promotes the development of the sprouts, but is ineffective in inducing changes in the RNA level when supplied to isolated explants of potato tuber both germinating or deprived of buds.

CHANGES IN THE ACTIVITY OF CHLOROGENIC ACID OXIDASE AND OTHER ENZYMES INVOLVED IN OXIDATION AND REDUCTION OF TPN IN AGEING POTATO TUBER SLICES. — The activation of respiration, and in particular of the pentose phosphate pathway, during incubation of potato tuber slices could depend on the increase of activity of oxidative enzymes mediating electron transfer from Gl. 6-P to oxygen. The present report deals with the activity changes, in the first period of incubation, of the following enzymes: Gl. 6-P-dehydrogenase, TPNH-glutathione reductase, gluta-thione-dehydroascorbate reductase, chlorogenic acid oxidase and a TPNH diaphorase utilizing tetrazolium salts as electron acceptors. The activity of all of these enzymes, with the exception of TPNH diaphorase, was found to bs, at all stages of incubation, in large excess respect that required to account for the estimated contribution of the pentose phosphate pathway to respiration. Gl. 6-P dehydrogenase, glutathione reductase and chlorogenic oxidase act...

The ultrastructural organization of mycelia of Neurospora crassa exponentially growing in different media has been studied. It can be seen that the diameter of the hyphae is larger in cells growing in richer media. More interesting large variations are found for several mitochondrial parameters: number of mitochondria per section, average mitochondrial area, and number of cristae per mitochondria. Unlike what is found in Saccharomyces cerevisiae , growth in sucrose does not repress the formation of mitochondria in Neurospora crassa , as can be seen by comparing the ultrastructures as well as the specific activity of cytochrome oxidase of the cells growing in sucrose or in acetate. It is suggested that this behaviour is correlated with the strictly aerobic respiratory metabolism of N. crassa . Further growth on rich media does not repress mitochondria formation.

The levels of magnesium and polyamines in mycelia grown in Vogel's minimal media supplemented with sucrose in the presence of both excess and growth-limiting concentrations of Mg 2+ have been determined. Exponentially growing mycelia in magnesium-rich media present the following average internal concentrations: 16 mM Mg 2+ , 16 mM spermidine, 0.22 mM spermine. A decrease in the cellular magnesium level induced in the mid-exponential phase is accompanied by a parallel increase in the polyamine levels, while growth and RNA content remain unaffected for several hours. In established magnesium starvation a decrease in the net synthesis of the RNA, and several hours later of DNA, occurs. The possibility that polyamines and magnesium may partially substitute for each other in vivo is discussed.

Particle weights of ribosomes and ribosomal subunits from Neurospora crassa have been calculated from the mol. wt of the rRNA and the relative RNA contents of the particles (as determined from buoyant density in CsCl). The high-salt washed monomeric ribosomes (3.95 · 10 6 –3.98 · 10 6 daltons) are identical in size to those of higher plants. Functionally active large and small subunits derived from “run-off” ribosomes of Neurospora exhibit molecular weights of 2.50 · 10 6 and 1.55 · 10 6 , similar to those isolated from several green plants. It is suggested that the sizes of both ribosomal subunits have been largely conserved in the plant kingdom, and that a change in the size of the large subunits has occurred only in animals, parallel with the increase in size of the major rRNA component.