Transpiration, a prerequisite for long-distance transport of minerals in plants?

The major "benefit" alleged to accrue from transpiration (the evaporative loss of water from plant surfaces) is that it is essential for the long-distance transport of mineral ions, but the possible interrelation between these two processes has rarely been tested. Transpiration was experimentally dissociated from mineral supply by growing sunflowers (Helianthus anuus) in hydroculture and providing mineral nutrients only during the nights. These plants grew as well as a control group that received nutrients only during the day and transpired 12-15 times more water during the exposure period. It thus appears that convective water transport in the xylem, brought about by root pressure and the resultant guttation, "growth water," and Münch's phloem counterflow is in itself sufficient for long-distance mineral supply and that transpiration is not required for this function.

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium.

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA.

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [(14)C]trioleoylglycerol, releasing [(14)C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [(14)C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [(14)C]oleic acid produced from the lipase reaction to [(14)C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.

An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants.

An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others.

Ca stimulated neutral lipase activity in castor bean lipid bodies.

The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca(2+) and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo.

Fatty acids of rice coleoptiles in air and anoxia.

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [(14)C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O(2), as it does in other plants.

ATPase in lipid body membranes of castor bean endosperm.

Lipid body membranes purified from castor seed endosperm of dry seeds and 4 d old seedlings were found to have an ATPase activity associated with them. This was confirmed by equilibrium density centrifugation of the membranes using acid lipase as a marker enzyme. The specific activity ranged from 45 to 200 nanomoles per milligram protein per minute. The pH optimum was 9.0 but at pH 7.5 nearly 40% of the maximum activity was retained. The apparent K(m) for Mg-ATP was 0.5 millimolar. A divalent cation was required for activity and Mg(2+) was the most effective. Other nucleoside triphosphates were also hydrolyzed but there was no hydrolysis of pyrophosphate or p-nitrophenylphosphate. The ATPase was not inhibited by oligomycin, vanadate, dicyclohexylcarbodiimide, or molybdate but was inhibited by sodium azide. Washing the membranes with increasing concentrations of NaCl removed up to 60% of the ATPase activity but none was removed by 3 millimolar ethylene-diaminetetraacetate.

Lectins in castor bean seedlings.

The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10-day seedlings that had been exposed to light from day 5. The embryonic axes of 2-day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture.The ability of proteinases and glycosidases (isolated from endosperm of 4-day seedlings) to hydrolyze the lectins was examined. No hydrolysis of the two lectins was observed, but the subunits, separated by reduction with 2-mercaptoethanol, were hydrolyzed slowly by a proteinase and some release of mannose was observed in the presence of the glycosidases. Ricin was converted to its subunits by cysteine and an enzyme in an endosperm extract accelerated chain separation by glutathione.

Purification and properties of glyoxysomal lipase from castor bean.

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings.

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P(2)) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P(2) is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P(2) was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P(2) in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P(2), respectively) were sufficient to account for the highest observed rates of Fru-2,6-P(2) metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P(2) level. Variation in the level of Fru-2,6-P(2) in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm.

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm.

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: effect on PPi:fructose 6-phosphate phosphotransferase.

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PPi:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PPi:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.

Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings.

During germination and early growth of castor bean (Ricinus communis), all cellular constituents of the endosperm are eventually transferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic acids. The total content of nucleic acids and nucleotides declines rapidly between day 4 and day 8 of seedling development. Concomitant with this decline, a secretion of adenosine, guanosine, and adenine from excised endosperms into the incubation medium takes place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could not be detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated metabolism. Uptake studies with cotyledons removed from the seedling showed that these have the ability to absorb all the substances released from the endosperm. Besides sucrose and amino acids, both nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP was not. Kinetic analyses were carried out to estimate the maximal uptake capacities of the cotyledons. Rates of uptake were linear for at least 1 to 2 hours and saturation kinetics were observed for all substances investigated. It is concluded that nucleosides can serve best as transport metabolites of nucleic acids, inasmuch as they are taken up by the cotyledons with the highest efficiency, the V(max)/K(m) ratios being considerably higher than those found for free purine and pyrimidine bases. For both adenosine and adenine transport, the V(max) was about 2 micromoles per hour per gram fresh weight, and the K(m) values were 0.12 and 0.37 millimolar, respectively. The rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo.

Transport of dicarboxylic acids in castor bean mitochondria.

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of (14)C from [(14)C]malate or [(14)C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of (14)C from [(14)C]malate or [(14)C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K(m) values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [(14)C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [(14)C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4 degrees C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [(14)C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K(m) of 2.5 millimolar and V(max) of 250 nanomoles per minute per milligram protein at 4 degrees C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

Alcohol dehydrogenase and an inactivator from rice seedlings.

Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N(2), ADH levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N(2) for 3 days, ADH levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N(2) led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N(2) appear to suggest a regulatory role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented.

Alcohol dehydrogenase inactivator from rice seedlings : properties and intracellular location.

The alcohol dehydrogenase (ADH) inactivator from aerobically grown rice (Oryza sativa) coleoptiles was shown to be associated with membranes which were recovered in sucrose gradients at peak density 1.13 grams per cubic centimeter. When Mg(2+) was included in the gradient, the inactivator was recovered at peak density 1.16 grams per cubic centimeter coinciding with the marker enzyme for endoplasmic reticulum, antimycin A-insensitive NADH cytochrome c reductase. ADH was recovered exclusively in cytosol fractions. The inactivator attacks ADH from several plant sources and from yeast. There was no evidence for proteolysis when pure yeast ADH was inactivated by the inactivator, but there was a loss of SH groups from ADH during inactivation which was restored after incubation with dithiothreitol under denaturing conditions. The inactivator did not attack other SH enzymes tested but did result in loss of SH groups from glutathione and dithiothreitol which prevent ADH inactivation. When O(2) was removed from the inactivator assay medium, the inactivation as well as the loss of SH groups from yeast ADH was significantly depressed.

Effects of o(2) concentration on rice seedlings.

The ability of rice, wheat, and oat seedlings to germinate and grow as the O(2) concentration was lowered to zero was compared. The germination of rice was completely unaffected by O(2) supply, whereas that of oats and wheat was strongly retarded at levels below 5% O(2). In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O(2) concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O(2), the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N(2), and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile.

Ricin inhibition of in vitro protein synthesis by plant ribosomes.

In vitro translation systems were prepared with supernatant factors from wheat germ and 80S ribosomes from wheat germ, barley embryos, watermelon cotyledons, pea cotyledons, and castor bean endosperm. Ricin A-chain, which strongly inhibits protein synthesis by mammalian ribosomes, inhibited all of the plant ribosomal systems by 50% when present at 25-45 mug/ml- approximately 23,000 times the concentration needed to inhibit mammalian systems. Ricinus communis agglutinin A-chain, a protein similar to ricin A-chain, inhibited translation by the plant systems 50% at concentrations 5-10 times those of the ricin A-chain. Ribosomes from castor bean endosperm, the source of ricin and the agglutinin, were just as susceptible to the inhibitors as were ribosomes from the other four plants. Compartmentation of the inhibitors within vacuoles derived from protein bodies of the endosperm appears to be responsible for protecting cytoplasmic protein synthesis during germination of castor beans.

Effects of leupeptin on proteinase and germination of castor beans.

Leupeptin, a tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloidstorage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.