Maltose uptake in the Zea mays scutellum

Maltose transport in slices of the maize scutellum was demonstrated despite the presence of an active maltase situated at the cell surface. The maltase could be inhibited or destroyed by treatments (neutral pH during uptake, pretreatment in Tris buffer at pH 7·5, or in 0·01 N HCl) that allowed appreciable rates of maltose uptake to occur. Using Tris- and HCl-treated slices, it was found that at disaccharide concentrations of 50 and 100 mM, maltose and sucrose were taken up at very nearly the same rates. At sugar concentrations below 50 mM, sucrose was taken up at greater rates than maltose. The maltose content of the slices was directly proportional to the maltose concentration of the bathing solution, and about 4 hr were required for equilibration. From this, it is concluded that one way maltose enters the slices is by free or facilitated diffusion. However, endogenous maltose is utilized by the slices at rates that are much too low to account for the net rates of maltose uptake. Although the slices contain a high level of surface maltase activity, only a low level of endogenous maltase activity was found. This probably accounts for the slow utilization of endogenous maltose. Therefore, the existence of a specific maltose transport system is proposed; a system that contains a carrier saturable with maltose, but one that does not release free maltose into the cytoplasm.     

Localization of a phosphatase (ATPase) on the plasmalemma of the maize scutellum

A phosphatase (ATPase) was demonstrated on the surface of the maize scutellum cell by showing that (1) when exogenous ATP was hydrolysed by intact scutellum cells, ADP, AMP and P_i appeared in the bathing solution in stoichiometric amounts, (2) the rate of hydrolysis was sensitive to bathing solution pH; (3) exogenous Mg²⁺ increased the rate of hydrolysis and (4) when the ATPase reaction was carried out in the presence of lead nitrate, TEM photographs showed lead phosphate deposits located almost exclusively in the plasmalemma. The ATPase was tightly bound to the plasmalemma and was not destroyed by freezing and thawing scutellum slices, a treatment which disrupted the plasmalemma. Acid treatment (10 mM HCl) of fresh or frozen-thawed scutellum slices destroyed acid phosphatase activity but had little effect on ATPase activity at pH 6.5. Following acid treatment of the scutellum slice preparations, a definite Mg²⁺ requirement for ATPase activity could be demonstrated.     

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.     

Sucrose efflux and export from the maize scutellum*

Abstract During incubation of maize scutellum slices in fructose, there was an efflux of sucrose. Efflux was constant for at least 4 h at fructose concentrations of 70 or 100 mol m^?3. Efflux was increased by EDTA, and decreased by Ca²⁺. Efflux was independent of pH after EDTA treatment, but increased from untreated slices when the pH was lowered from 7 to 4. Uranyl ion and PCMBS (p-chloro-mercuribenzenesulfonic acid) abolished sucrose uptake, but were only weak inhibitors of sucrose efflux. These results are consistent with efflux occurring by simple diffusion through aqueous pores, but they do not rule out facilitated diffusion. Rates of sucrose export from the scutellum to the root shoot axis were estimated from measurements of axis respiration and dry weight gain. Sucrose efflux from scutellum slices was only 14-22% of the export rate. Sucrose efflux from the whole scutellum was only 3-4% of the export rate. It is concluded that the observed efflux is from leaky cells and does not represent sucrose on the way to the phloem along a path that includes the apoplast. These results support the idea that the path for sucrose from parenchyma cell to sieve tube in the maize scutellum is entirely symplastic.     

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.     

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.     

Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.     

Sugar transport in the light leaf spot pathogen Pyrenopeziza brassicae

Abstract In the light leaf spot fungus Pyrenopeziza brassicae , the kinetics of uptake of sucrose, glucose and fructose are all biphasic. At low and high concentrations, glucose and fructose share a high-affinity and a low-affinity hexose carrier respectively, with K _m s of 3.5 and 4.6 μM for uptake of glucose and fructose respectively by the high-affinity system, and K _s s of 690 and 750 uM for uptake of glucose and fructose by the low-affinity system. The data also suggest the existence of separate high-affinity and low-affinity uptake systems for sucrose. P. brassicae possesses both soluble and paniculate invertase activity, with pH optima of 5.0 and 4.0 respectively. Activity of the particulate invertase is considerably in excess of the highest rates of sucrose uptake.     

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.     

Glucose control of sucrose synthase in the maize scutellum

The in vivo amounts of UDPG, UTP, UDP and UMP, metabolites known to influence the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS), were measured throughout 5 hr incubations of scutellum slices in fructose or water, i.e. under conditions of sucrose synthesis or breakdown. Cytosolic concentrations were estimated assuming that these metabolites were confined to the cytosol. Within the estimated in vivo concentration ranges, UDPG, UTP and UDP had little effect on the in vitro SS activity, but glucose (100 mM) inhibited SS in the synthesis direction by 63–70% and in the breakdown direction by 86–93%. Glucose inhibition of SS was considerably less when saturating levels of substrates were used. Sucrose did not inhibit SS. It is concluded that during germination the glucose produced from starch breakdown in the maize endosperm enters the scutellum and inhibits SS, preventing a futile cycle and limiting SS participation in sucrose synthesis.     

Accumulation and conversion of sugars by developing wheat grains. 3. Non-diffusional uptake of sucrose, the substrate preferred by endosperm slices

Abstract. Starch synthesis by developing wheat endosperm slices incubated in liquid media was more rapid, at optimum concentration, from sucrose as external substrate than from glucose and/or fructose. Fructose inhibited conversion of sucrose or glucose. The results are consistent with the hypothesis that sucrose is not hydrolysed in the apoplast before uptake.
Besides a diffusional influx and efflux of labelled sucrose there was a non-diffusional influx; it was inhibited by dinitrophenol, potassium arsenate, potassium iodide, and parachloromercuribenzene sulphonate (PCMBS). PCMBS inhibited both uptake and conversion of label from 150 molm⁻³¹⁴C-sucrose by 75%. Uptake and conversion of sucrose were stimulated by lowering pH and by fusicoccin, a promoter of proton extrusion.
Extracellular solutes like raffinosc and polyethylene glycol stimulated net uptake of label from ¹⁴C-sucrose — the larger molecule being more effective — this being due to a non-specific inhibition of diffusional efflux. At too high an osmotic concentration such solutes reduced net uptake; the larger the molecule the lower this transitional concentration.
In conclusion, wheat endosperm is better equipped to convert apoplastic sucrose rather than the hydrolysis products to starch; active loading of sucrose possibly involves proton co-transport; and large molecules in the extracellular solution reduce the diffusional elllux of loaded substrate.     

Dinitrophenol-induced efflux of sucrose from maize scutellum cells

Maize scutellum slices accumulated sucrose during incubation in glucose, fructose or sucrose. Sucrose was accumulated in two compartments, tentatively     

Isolation and characterization of a cDNA clone from Lolium temulentum L. encoding for a sucrose hydrolytic enzyme which shows alkaline/neutral invertase activity

A novel cDNA clone, functionally expressed in E. coli, was isolated from a L. temulentum L. cDNA library. The expressed protein hydrolysed sucrose with an apparent Km of approximately 18 mM, and produced equi-molar concentrations of glucose and fructose. Optimum activity was observed at pH 7-7.5; there was little or no activity at pH 5.5. The expressed protein did not hydrolyse raffinose, stachyose or maltose. The activity of the expressed protein was inhibited by fructose (50% at 15 mM) and TRIS (50% at 2.5 mM), but was not affected by MgCl2, CaCl2 or MnCl2. These findings suggest that this cDNA clone encodes for an alkaline/neutral invertase. Sequence analysis revealed little homology with published sequences for acid invertase, however the invertase motif (NDPN) identified in other invertases was present. Expression studies show that the gene encoding for this enzyme is not regulated by sucrose accumulation in leaf tissue.     

Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick

1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different K(m) values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose, palatinose and maltose. The second peak contained three disaccharidase enzymes all specific for the hydrolysis of maltose. 8. It is proposed that the intestinal disaccharidases of the chick exist in the form of two complexes: a sucrase-isomaltase complex and a maltase complex.     

Production of alpha-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (相似文献   

Resistance to quaternary ammonium compounds in Staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pST827

K.A. ALBASHERI AND W.J. MITCHELL. 1995. Maltose metabolism in the obligate anaerobe Clostridium acetobutylicum was studied. The sugar is accumulated via an energy-dependent transport process which is not a phosphotransferase. Cell extracts were incapable of phosphorylating maltose in the presence or absence of phosphoenolpyruvate or ATP, but exhibited hydrolytic activity against a range of glucoside substrates. The activity was predominantly in the soluble fraction of cell extracts, indicating a cytoplasmic location in the cell. Gel filtration on Sephadex G100 indicated the presence of at least two α-glucosidases. One enzyme (maltase) was active with maltose and maltotriose, while the other (pNPGase) hydrolysed isomaltose and several glucoside analogues, but neither showed activity against starch. Both glucosidases were induced by isomaltose, maltose, glucose and starch, but not by xylose, sucrose or cellobiose. In the presence of both glucose and maltose, growing cells showed a preference for glucose, apparently due to regulation of maltose transport, which did not occur in glucose-grown cells.     

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     

Intestinal disaccharidase activities in the chick

1. Disaccharidase activities of the small and large intestines of the chick were studied. 2. Homogenates of the small intestine readily hydrolysed maltose, sucrose and palatinose (6-O-α-d-glucopyranosyl-d-fructose), hydrolysed lactose slowly and did not hydrolyse trehalose and cellobiose. 3. Within the small intestine the disaccharidases were located mainly in the intestinal wall; the activity in the contents accounted for less than 5% of the total activity. 4. The disaccharidases were non-uniformly distributed along the small intestine, the activities being greatest in the middle section. 5. The disaccharidase activities increased with age between 1 and 43 days. 6. Homogenates of the large intestine and contents readily hydrolysed maltose, sucrose, palatinose and lactose and hydrolysed cellobiose and trehalose slowly. 7. The large-intestinal disaccharidases were located mainly in the contents. 8. Similar K_m and pH optimum values were found for the maltase, sucrase and palatinase activities of the large and small intestines. 9. The lactase activity of the large intestine was markedly affected by diet and had different K_m and pH values from the small intestinal lactase. 10. Low activities of intestinal disaccharidase were found in 12-day-old embryos and marked increases in the intestinal disaccharidases of the developing embryo occurred 2–3 days before hatching.