Invertase and maltase in the free space of the maize scutellum

When maize scutellum slices were incubated in solutions of sucrose or maltose, there was a release of glucose into the bathing solution. The pH optima for glucose release were 2.5 for sucrose and 3.5 for maltose. From measurement of rates of glucose uptake into slices in the presence or absence of sucrose, it is calculated that glucose uptake will introduce errors of 3–9%, depending on the sucrose concentration, in estimates of free-space sucrose-hydrolase activity at pH 2.5. At their respective pH optima, maltose was hydrolysed at a rate 2.5 times that of sucrose. When frozen-thawed slices were used the same pH optima were obtained, but rates of hydrolysis were increased. Raffinose and melezitose also were hydrolysed with pH optima of 2.5 and 3.5, respectively. α-Methyl glucose was not hydrolysed. A 60-min HCl treatment (pH 2) of scutellum slices destroyed 69% of the sucrose-hydrolase activity and 100% of the maltose-hydrolase activity. In contrast, sucrose uptake and sucrose synthesis from exogenous fructose were not affected by HCl treatment. It is concluded that there are two hydrolases, acid invertase and maltase; that they are either on or outside the plasmalemma (in the free space); and that they are not necessary to the disaccharide uptake processes either by supplying exogenous hexose or by acting as transporters.     

Sucrose transport and hexose release in the maize scutellum

Since hexoses readily diffuse from maize scutellum cells, it should be possible to detect them if they are produced during sucrose transport at the tonoplast or the plasmalemma. To test this idea, scutellum slices were placed in dinitrophenol (DNP) (which inhibits hexose utilization while greatly increasing utilization of vacuolar sucrose), and the utilization, uptake and leakage of sugars were measured. Only negligible amounts of hexose appeared in the DNP solution during a 5-hr incubation during which the slices metabolized 72μmol of sucrose. Glucose and fructose, added at a concentration of 2 mM, were taken up by the slices at rates 33% and 14% (respectively) of the rate of vacuolar sucrose utilization. It is suggested, therefore, that sucrose transport at the tonoplast does not release free hexose into the cytoplasm. Sucrose transport at the plasmalemma was studied using DNP- and mannose-treated slices. During incubation of these slices in sucrose, the disappearance of sucrose resulted in the appearance of significant quantities of glucose and fructose in the bathing solution. Evidence is presented that sucrose is split into glucose and fructose during transport across the plasmalemma. It is concluded that free hexose is not normally a product of this splitting but is a result of an uncoupling in the transport system caused by the DNP or mannose treatments.     

Further study of the Hansenula polymorpha MAL locus: characterization of the alpha-glucoside permease encoded by the HpMAL2 gene

The HpMAL2 gene of the MAL gene cluster of Hansenula polymorpha codes for a permease similar to yeast maltose and alpha-glucoside transporters. Genomic disruption of HpMAL2 resulted in an inability of cells to grow on maltose, sucrose, trehalose, maltotriose and turanose, as well as a lack of p-nitrophenyl-alpha-D-glucopyranoside (PNPG) transport. PNPG uptake was competitively inhibited by all these substrates, with Ki values between 0.23 and 1.47 mM. Transport by HpMal2p was sensitive to pH and a protonophore carbonyl cyanide-m-chlorophenylhydrazone (CCCP), revealing its energization by the proton gradient over the cell membrane. Although HpMAL2 was responsible for trehalose uptake, its expression was not induced during trehalose growth. A maltase disruption mutant did not grow on maltotriose and turanose, whereas it showed normal growth on trehalose, demonstrating the dispensability of maltase for intracellular hydrolysis of trehalose. In a Genolevures clone pBB0AA011B12, the promoter region and the N-terminal fragment of the putative transactivator of MAL genes is located adjacent to HpMAL2. A reporter gene assay showed that expression from that promoter was induced by maltose and sucrose, repressed by glucose, and derepressed during glycerol and trehalose growth. Therefore, we presume that the gene encodes a functional regulator.     

Transport and hydrolysis of disaccharides by Trichosporon cutaneum.

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 +/- 3 mM sucrose or lactose and 5 +/- 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, K+, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis.

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

Factors Affecting Glucose and Maltose Phosphorylation by the Ruminal Bacterium Megasphaera elsdenii

The objectives of this study were to examine the effects of growth substrate and extracellular pH on phosphoenolpyruvate-dependent glucose phosphorylation as well as to examine how maltose is phosphorylated by the ruminal bacterium Megasphaera elsdenii B159. Phosphoenolpyruvate-dependent glucose phosphorylation by toluene-treated cells was constitutive, and glucose phosphorylation was reduced by 69% at pH 5.0. When toluene-treated cells were incubated in histidine buffer, little maltose phosphorylation occurred in the absence of inorganic phosphate. However, the addition of increasing concentrations of either potassium or sodium phosphate increased maltose phosphorylation. Maximal phosphorylation activity was observed at between 25 and 50 mM of either inorganic phosphate source. Compared with the control incubations, maltose phosphorylation was increased over threefold with 25 mM of either potassium or sodium phosphate. Phosphoglucomutase activity was detected in cell extracts of M. elsdenii B159, and this enzyme had a K _m of 3.2 mM for glucose-1-P and a V _max of 1836 nmol of NADP⁺ reduced/mg of protein per min. Maltose was also hydrolyzed by an inducible maltase (K _m , 1.19 mM). To our knowledge, this is the first report of a maltose phosphorylase and a maltase in M. elsdenii. Received: 3 November 1999 / Accepted: 5 January 2000     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase-glucoamylase identical with membrane-bound maltase-glucoamylase in molecular weight, heat-sensitivity, substrate specificity, K(m) for maltose and K(i) for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase-glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its K(m) for maltose was 1.5mm. It was inhibited by turanose (K(i)=7.5mm) and Tris (K(i)=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50 degrees C for 10min. The acid maltase closely resembled beta-glucuronidase and acid beta-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.     

Investigations into the Role of Sucrose in Potato cv. Estima Microtuber Production in vitro

Sucrose has been the carbohydrate traditionally used for potatomicrotuber production. Added to nutrient media, sucrose canact solely as a carbon source, or as an osmoticum, or both.Preliminary tests showed that the osmolarity of sucrose solutionswas increased by autoclaving, indicating some breakdown of thesugar. This was taken into consideration in experiments whichinvolved supplementing 4% sucrose media with sucrose, maltose,glucose or fructose, while keeping the osmotic potential ofthe media constant. A medium concentration of about 400 mM withonly sucrose was more suitable for microtuber production thanmedia supplemented with maltose, glucose or fructose. However,a better microtuber yield was obtained when hexoses were addedthan with unsupplemented 4% sucrose media. When glucose wassupplied at concentrations which had the same number of carbonatoms as 8% sucrose, the high osmolarity inhibited microtuberisation.Sugar movement in the tubering plantlet was followed using radio-labelledsucrose, glucose and fructose. The sucrose was translocatedand used at a faster rate than the other sugars, which tendedto remain in the roots of the plantlets. Furthermore, therewas no difference in microtuber production on media to whichthe sucrose was added before or after autoclaving, indicatingthat levels of breakdown were not severe enough to affect theprocess. Therefore, it is concluded that sucrose acts primarilyas a suitable carbon source for uptake and utilization by theplantlets, but, at 8%, it also provides a favourable osmolarityfor the development of microtubers.Copyright 1995, 1999 AcademicPress Solanum tuberosum (L.), potato, microtuber, media, sugar, sucrose, osmolarity, pH     

Extracellular Maltase of Bacillus brevis

Bacillus brevis NRRL B-4389 produced extracellular maltase (alpha-glucosidase; EC 3.2.1.20) only in the presence of short alpha-1,4-glucosidic polymers, such as maltose and maltotriose. An optimum medium was developed; it contained 2.5% maltose, 0.5% nonfat dry milk, 0.4% yeast extract, and 0.01% CaCl(2). The enzyme was produced extracellularly during the logarithmic phase of growth; no cell-bound activity was detected at any time. Partial purification of the maltase was accomplished by using diethylaminoethyl cellulose batch adsorption, ammonium sulfate precipitation, and Sephadex G-200 gel filtration. Maltase, isomaltase (oligo-1,6-glucosidase), and glucosyltransferase activities were purified 20.0-, 19.1-, and 11.5-fold, respectively. Some properties of the partially purified maltase were determined: optimum pH, 6.5; optimum temperature, 48 to 50 degrees C; pH stability range, 5.0 to 7.0; temperature stability range, 0 to 50 degrees C; isoelectric point, pH 5.2; and molecular weight, 52,000. The relative rates of hydrolysis of maltose (G(2)), maltotriose (G(3)), G(4), methyl-alpha-d-maltoside, G(40), dextrin, and isomaltose were 100, 22, 12, 10, 10, 8, and 5%, respectively; the K(m) on maltose was 5.8 mM; d-glucose, p-nitrophenyl-alpha-d-glucoside, and tris (hydroxymethyl) aminomethane were competitive inhibitors; transglucosylase activity of the enzyme on maltose resulted in the synthesis of isomaltose, isomaltotroise, and larger oligosaccharides.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

Sucrose Transport in Isolated Immature Barley Embryos

Techniques have been devised to select immature barley embryosat various stages in their development, and to study their accumulationof sucrose in vitro. Isolated embryos accumulate sucrose overa period of several hours of which some 80 per cent is conservedas a pool of free sucrose and the remainder utilized in macromolecularsynthesis. The rate of sucrose uptake increases with embryodevelopment, however the specific activity of uptake remainsconstant, indicating that the transport processes are fullyoperative early in embryogenesis. From the kinetics of sucroseuptake it is deduced that facilitated transport predominatesat sucrose concentrations of 50 mM, while at higher concentrationspassive diffusion makes an increasing contribution to sucroseaccumulation. The substrate specificity and the sensitivityof sucrose transport to uncoupling agents, in addition to thestability of the pool of accumulated sucrose, are all indicativeof active transport playing a major role in the sucrose assimilationof developing barley embryos. Hordeum distichum (L.) Lam, barley, embryo, sucrose transport