Attività Respiratoria Livelli Enzimatici e Tenore in Acqua Durante la Maturazione del Seme di Ricino

Abstract

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.     

Variazioni di Metaboliti Nel Seme di Ricino in Fase di Maturazione

Abstract

Changes of glycolytic substrates level during ripening of the castor bean seed. — The changes of the concentration of carbohydrates and of the main glycolytic substrates in the castor bean seed during the ripening phase were investigated. The following results were obtained:

The level of unphosphorylated sugars and of acid hydrolysable polysaccharides remains almost unchanged, with a tendency to a rise during the ripening phase. The slight increase of these compounds, together with the transition of the R. Q. from high to low values, might be interpreted as an indication of a shift of the seed from the a metabolism of fat synthesis to one of conversion of lipids into sugars, such as is observed in the germinating castor bean seeds.

Hexose monophosphate level sharply decreases during the last period of maturation. However, the level of these substrates does not fall so low as to suggest a severe limitation for the pentose-P pathway activity.

Fructose diphosphate, DOAP, GAP, 3 PGA, 2 PGA, PEP and pyruvate levels consistently increase during the ripening process. This indicates that the drop of oxygen uptake observed in this phase cannot be due to a lack of glycolytic substrates. On the other hand, the ratios between some substrates are shifted, during ripening, from values close to the theoretical equilibrium constants to quite different values. This finding, when correlated with the one of the strong decrease of the glycolytic flow, strongly suggests a severe inactivation of the glycolyting enzymes during ripening.

The increase of pyruvate in tissues showing a decreasing respiratory activity indicates a fall of the oxidative capacity of mitochondria. This might be due to a lack of ADP, or other high energy bond acceptor, following a block of synthetic processes. However, no decrease of ADP level, and an increase of the ADP/ATP ratio during ripening is observed, Among the alternative hypothesis: a) lack or excess of oxalacetate; b) increase of concentration of some Krebs cycle inhibitor; c) inactivation of mitochondrial enzymes, the latter is thought most probable, in view of the finding of a sharp decrease of some other enzyme activities during ripening, of the above mentioned interpretation of the shift of the ratios between glycolytic substrates, and of the very low level of mitochondrial activity in preparation from the mature castor bean seed. These results when correlated with those from parallel investigations on the biochemistry of castor bean seed maturation and germination suggest, as a working hypothesis, that the respiratory metabolic inactivation accompanyng seed repening is due to a general block of the metabolism of ribonucleic acid and thus protein synthesis.     

Aspetti Enzimatici Della Maturazione e Germinazione dei Semi di Ricino

Abstract

Enzyme levels during ripening and germination of castor bean seeds. — During the development of the endosperm of castor bean seeds two distinct phases can be recognized: pre-maturation and germination. The former is characterized, metabolically, by the rapid conversion of carbohydrates into lipids, and storage proteins. The latter is characterized by the reconversion of these storage materials into sugars. Both these processes are dependent upon the activity of the glycolytic pathway; for this reason the behaviour of some enzymes of this pathway and, in general, of the carbohydrate metabolism have been studied during the two phases. The changes (during the evolution of the seeds) of the following enzymes have been studied:

Gl-6-P-dehydrogenase, 6-P-gluconate dehydrogenase, P-glucomutase, Hexokinase Hexoseisomerase, Aldolase, alcaline and acid Phosphatase, Pyrophosphatase and ATP-ase.

All these activities have been measured in the 20.000 × g supernatant fraction of cell homogenates.

The results show that all the enzymes activities measured increase rapidly during the period of accumulation of storage materials. In the following period all of these activities decrease until the stage of ripeness of the seed. During the first few days of germination the activities increase again rapidly. A particular behaviour is the one of Fr-1-6-P-phosphatase (the enzyme cleaving the phosphate bond in C ¹ position). This enzyme reaches during germination a level much higher than the maximal observed during the ripening process. This might be an important fact correlated with the inversion of the glycolytic reactions during germination.     

Effetto di Inibitori Della Sintesi Proteica Sull'aumento di Attività Enzimatiche e sul Risveglio del Metabolismo Nell'Endosperma di Semi di Ricino in Germinazione

Abstract

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.     

Sull'evoluzione Dei Mitocondri Nell'endosperma di Semi di Ricino in Germinazione

Abstract

On the behavior of mitochondria in the castor bean seed endosperm during the early phases of germination. — In the endosperm of the castor bean seed the oxidative activity and the protein nitrogen contents of the mitochondrial fraction markedly increase during the first period of germination (Beevers and coworkers). The activation of the mitochondrial system is paralleled by a similar increase of the activity of several soluble enzymes; the latter process is severely depressed by protein synthesis inhibitors (Cornaggia, Aberghina).

The present research is aimed to understand at what extent phenomena of activation and/or, respectively, of « ex novo » synthesis are responsible of the increase of mitochondrial activity. The following aspects of the mitochondrial behavior during the early period of germination were investigated:

a) Changes in the activity of cytochrome oxydase, malate dehydrogenase and of the succinate-citochrome reductase system.

b) Changes in the morphology of mitochondria and other particulated cell structures, as revealed by electron microscopy.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

These results, integrated with those of other investigations on the same material are in agreement with the hypothesis that the activation of metabolism in the endosperm during germination depends in a very early phase mainly on the transition of enzyme systems from an inactive to an active state; while in a second phase synthesis « ex novo » of enzymes and cell structures predominates.     

Transfer of phospholipids from microsomes to mitochondria in germinating castor bean endosperm

¹⁴C-labeled microsomes were prepared by feeding [1-¹⁴ C]acetateto endosperm tissues from 4-day-old seedlings of castor beanseeds and incubated with unlabeled mitochondria from the sametissues. The loss of ¹⁴C-lipids from the microsomes was accompaniedby an increase of ¹⁴C-lipids in the mitochondria. The additionof 105,000?g supernatant and also pH 5.1-treated supernatant,both of which had been prepared from castor bean endospermsat the same stage, markedly enhanced the lipid transfer frommicrosomes to mitochondria. The activity in this fraction wasprecipitated by ammonium sulfate and lost with trypsin or heattreatment. The transfer of lipids was limited to phospholipids.Thus, it is concluded that in castor bean endosperms, phospholipidsare transferred from the endoplasmic reticulum to the mitochondriaby a phospholipid-exchange protein contained in the cytosol. (Received August 8, 1977; )     

Evoluzione Del Metabolismo Ossidativo Dei Semi di Ricino Durante la Maturazione

Abstract

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. suggests that the oxidative activity of these seeds in the last stages of maturation is supported either by the oxidation of substrates more reduced than carbohydrates (probabily fats), or, partially, by the conversion of fats to sugars.     

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p-nitroaniline from the hydrolysis of benzoyl-l-Asn-p-nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]-napthy])-ethylenediamine dihydrochloride: diazotized p-nitroaniline was determined spectrophotometrically at 548 nm (?₅₄₈= 1.64 × 10^?1M^?1 cm^?2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH-activity profiles with optimal activity occuring at pH 5.4: 2) are heat-labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2-fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2-day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.     

Rapporti Tra Assunzione D'acqua,Metabolismo e Sintesi di Enzimi Nell'endosperma Di Semi Germinanti di Ricinus Communis

Abstract

Water uptake, activation of metabolism and enzyme synthesis in germinating castor bean seeds. — During the first days of germination water uptake by the castor bean seed endosperm is accompanied by a rapid rise of respiratory activity and of the « in vitro » detectable activity of a number of enzymes. The finding that the increase of enzyme activity is strongly inibited by protein synthesis inhibitors suggests an « ex novo » synthesis of enzymes in the endosperm of the germinating seed. The present investigation on the relationship between water uptake, metabolic activity and enzyme activity level lead to the following conclusions:

I - The increase of enzyme activity is strictly dependent on the availability of water, and on the rate of water uptake. When water uptake is depressed by incubation of the seed in high osmolarity media, enzyme activation is also severely depressed.

This is also observed when the seeds are germinating in contact with an amount of water consistently lower then the one they would taken up, in a given time (24 h), under conditions of unlimeted water availability.

II - The temperature coefficient of water uptake is close to 1.5 during the first 24 h, higher than 2 in the following 3 days. Low temperature almost completely inhibits the increase of enzyme activities in the endosperm.

III - Anaerobiosis inhibits the rate of water uptake by about 50%, in the first 24 h, and almost completely, in the following 3 days. Also the rise of enzyme activities is severely inhibited by lack of oxygen. The effect of protein synthesis inhibitors on water uptake is somewhat smaller, and the one on enzyme activity is somewhat larger than that of anaerobiosis.

These results are interpreted as indicating that during the early period of germination water uptake becomes more and more dependent on the metabolic activities of the endosperm cells, in as much the latter lead to the appearance of osmotically active substances and, possibly, to changes of the cell wall properties.

On the other hand, the level of hydration of the cytoplasm represents a limiting factor for the development of the mechanism involved in enzyme synthesis and metabolic activation.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). Received: 20 December 1997 / Accepted: 18 March 1998     

Osservazioni Preliminari sui Sistemi Degradanti dell'RNA nei semi di Ricino

Abstract

Preliminary observations on the enzymatic degradation of RNA in castor bean seeds. — Cocucci, Maggio, Monroy and Marrè have shown the decrease of RNA content during ripening in castor bean seeds, and its increase during germination. Furthermore, these Authors have demonstrated that in the dry ripe seeds the ribosomes are undetectable, and that they increase rapidly during germination. Two peaks of ribosomes are easily detected upon ultracentrifugal analysis in germinating seeds (Cocucci and Sturani). These observations were the basis for our investigations of the enzymes of RNA metabolism in castor bean seeds. This paper deals with our preliminary observations on RNA degrading enzymes in these tissues. We have been able to measure RNase activity, phosphodiesterase, 3′-,5′- and 2′-nucleotidases in castor bean seeds at different stages of development. RNase activity (measured in crude extracts) changes little during the ripening process, its rate corresponding to 40–50 μMoles of nucleotides liberated from RNA per hour and per gram of fresh weight. In the dry seeds, RNase activity is 30–40 μMoles of nucleotides/h.g.f.w., and it increases to about 60–70 μMoles/h/g.f.w. after 72 hours of germination.

Phosphodiesterase activity is about 4–5 μMoles/h.g.f.w.

The following rates have been found in seeds almost completely ripe seeds for 3′-, 5′- and 2′-nucleotidase activities, respectively 45–50 μMoles/h.g.f.w.; 6–7 μMoles/h.g.f.w.; 8 μMoles/h.g.f.w.; ATP-ase activity was of about 80–100 μMoles of phosphate liberated /h.g.f.w. - The high activity of 3′-nucleotidase, of the same order of that of RNase, suggests that these two enzymes are responsible for degradation of RNA to nucleosides and inorganic phosphate. Further investigations are being carried on to define the biochemical properties of castor bean RN-ase.     

Fenomeni di Inattivazione e di Riattivazione Enzimatica in Endospermi di semi di Ricino

Abstract

Inactivation and riactivation of enzymes in endosperms of castor bean seeds. — On the basis of previous results, the possibility has been investigated of the reversible interconversion of active and inactive form of enzymes in castor bean seeds, during their development.

The results described here indicate that:

1. the activity of some glycolytic enzymes increases greatly (81% and 400% increase of, respectively, Gl-6-P-dehydrogenase and aldolase) upon incubation of dry seeds for few hours at 4 °C.

2. The decrease of enzyme activity upon dehydration of seeds and the increase during the subsequent imbibition can be shown reproducibly.

3. This same observation is made for oxygen uptake.

These results are interpreted to indicate the reversible inactivation of enzymes caused by dehydration of seeds.     

Nuove Prospettive Nello Studio dei Fattori che Controllano la Germinazione Dei Semi

Abstract

New perspectives in the study of factors which control seed germination. — Seedlings of Triticum durum, cv. « Cappelli », coming from unirradiated embryos grafted on to irradiated endosperms (EM(u)/EN(i) of presoaked seeds (in distilled water for 24 h. at 20[ddot]C.) (treatment: X-rays, doses, 2,4,6,8,10 and 20 Kr), grow more than seedlings of control EM(u)/EN(u) (dose 0) (fig. 1). To have this reaction, it is necessary that the used seeds be after-ripe; at the various stages of seed ripening, be ginning from the milk stage, the phenomenon is not present.

On this basis, the author has thought that a natural inhibitor occurs in the after-ripe endosperm of « Cappelli », which is neutralized or destroyed by X-rays.

As an experimental demonstration, some trials have been made of growing wheat seedlings in Petri dish, on moistened (distilled water) filter paper, together with excised embryos or isolated endosperms (fig. 2): the after-ripe endosperm is able to depress the seedling growth (fig. 3). In the same experimental conditions, X-rays, dose 6 Kr, neutralize the inhibition effect given by the endosperm. (fig. 4).

A completely different situation occurs in wheat seed, during its ripening: endosperm is inactive, embryo produces inhibition effects on the seedling growth, which, also in this case, are reduced by X-rays.

These phenomena, put in relation with dormancy in Triticum durum, cv. « Cappelli », which is a relative dormancy, having its maximum at the milk stage, have led the author to the general conclusion that, during dormancy, a germination inhibitor occurs in the embryo of wheat seed; when dormancy is finished, the inhibitor appears in the endosperm, in a situation which becomes stable and definitive.     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

Ricerche Sulla Flora e Vegetazione dei Fontanili Dell'Agro Milanese

Abstract

Nucleic Acids in Ripening of Castor Bean Endosperm. - I. Quantitative and Qualitative Changes of total RNA. — The ripe Castor Bean seed endosperm (Ricinus Communis var. Sanguinea) has a very low level of nucleic acids and particularly of ribosomal RNA. However, they rapidly increase during the germination of the seed (S. Cocucci et al., Acc. Naz. Lincei, 38, 545, 1965).

The behaviour of the nucleic acids, obtained by phenol extraction from Castor Bean seed endosperm at different ripening stages, is the following:

1. From the moment when the developing seed has reached the maximal fresh weight to the moment of its complete ripening, the RNA content decreases from about 700 μg per seed to about 200 μg.

2. During the same period the DNA level (about 13–15 μg per seed) remains nearly costant.

3. Fractionation by sucrose density gradient of the phenol extracted RNA (H. L. Sanger and C. A. Knight, Biochem. Biophys. Reserch Commun., 13, 445, 1963) shows that during the ripening of the seed the high molecular weight RNA decreases much more than the low molecular weight RNA.

4. Radioactive phosphate injected in the seeds is incorporated in the RNA at all the stages. Fractionation by sucrose density giadient shows that the phosphate is essentially incorporated in low molecular weight RNA and there is pratically no labelling of the heavier RNA after quite a long period (2 hr.).

These data suggest that the decrease of the RNA during the ripening of the seed can be due to a fall in the ability to synthesize high molecular RNA.     

Inactivation of the glyoxysomal citrate synthase from castor bean endosperm by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)

Summary Two isoenzymes of citrate synthase were found in the endosperm of germinating castor bean seeds. One isoenzyme is restricted to mitochondria and the other to glyoxysomes. The two citrate synthases can be separated by (NH₄)₂SO₄ gradient solubilization, eluting at 58 and 43% (NH₄)₂SO₄, respectively. They are easily distinguished by the sensitivity to 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of oxalacetate: the glyoxysomal enzyme is completely inactivated within 15 seconds, while the mitochondrial enzyme remains unaffected. The time course of inactivation is a first order reaction. Oxalacetate prevents inactivation in high concentrations. The differences in DTNB sensitivity of the two citrate synthases can, in turn, easily be used to distinguish between the two isoenzymes. Since DTNB is a chromogenic compound in the assay for citrate synthase, it interfers with the assay at low concentrations of oxalacetate during K_m determinations. This can be avoided by other assays which do not include DTNB. The inactivation of the glyoxysomal citrate synthase of castor bean endosperm is similar to the known inactivation of prokaryotic citrate synthases.Abbreviation DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.