The effect of the seed coat, embryonic axis and aeration conditions on starch degradation hi cotyledons of Lens culinaris

The effect of exogenously applied galactose on the cell wall polysaccharide synthesis and UDP-sugar levels in oat ( Avena sativa L. cv. Victory I) coleoptile segments was studied to clarify the mechanism of inhibition of IAA-induced cell elongation by galactose, and the following results were obtained: (1) The inhibition of IAA-induced cell elongation by galactose became apparent after a 2 h-lag, while the lag was shortened to 1 h when galactose was added to the segments after more than 1 h of IAA application. (2) Galactose inhibited the [¹⁴C]-glucose incorporation into cellulosic and non-cellulosic fractions of the cell wall and the increase in net polysaccharide content in the fractions during long-term incubation. (3) The dominant sugar nucleotide in oat coleoptiles was UDP-glucose (2.1 nmol segment⁻¹). Galactose application caused a remarkable decrease in the UDP-glucose level, accompanying a strong accumulation of galactose-1-phosphate and UDP-galactose. (4) Galactose-1-phosphate competitively inhibited the UTP: a- d -glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) activity of the crude enzyme preparation from oat coleoptiles. From these results we conclude that galactose inhibits the IAA-induced cell elongation by inhibiting the formation of UDP-glucose, which is a key intermediate of cell wall polysaccharide synthesis.     

Cell wall modifications during auxin-induced cell extension in monocotyledonous and dicotyledonous plants

There are several differences between monocotyledonous and dicotyledonous plants. The sensitivity towards added galactose which inhibits auxin-induced coleoptile elongation but not stem elongation is one of the conspicuous differences between the two types of plants. InAvena coleoptile segments, galactose, probably as galactose-1-phosphate, inhibits the formation of UDP-glucose from glucose-l-phosphate. The inhibition of UDP-glucose formation due to galactose is not found inPisum epicotyl segments. InAvena UTP: α-D-glucose-1-phosphate uridyltransferase (EC 2.7.7.9) which catalyzes the reaction from glucose-1-phosphate to UDP-glucose seems to be inhibited by galactose-1-phosphate.     

Galactose prevents proton excretion but not IAA-induced growth in segments of azuki bean epicotyls

We investigated the effect of galactose on IAA-induced elongation and proton excretion in azuki bean (Vigna angularis Ohwi et Ohashi) segments in order to confirm whether or not protons were involved in auxin-induced growth. Galactose inhibited the IAA-induced decrease in the solution pH but had no inhibitory effect on IAA-induced growth in segments of azuki bean epicotyls. On the other hand, galactose inhibited both IAA-induced growth and proton excretion in oat (Avena sativa L.) coleoptile segments. From these results it is unlikely that IAA-induced growth is mediated by proton excretion at least in azuki bean epicotyls.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

Galactose Expulsion during Lactose Metabolism in Lactococcus lactis subsp. cremoris FD1 Due to Dephosphorylation of Intracellular Galactose 6-Phosphate

In Lactococcus lactis subsp. cremoris FD1, galactose and lactose are both transported and phosphorylated by phosphotransferase systems. Lactose 6-phosphate (lactose-6P) is hydrolyzed intracellularly to galactose-6P and glucose. Glucose enters glycolysis as glucose-6P, whereas galactose-6P is metabolized via the tagatose-6P pathway and enters glycolysis at the tagatose diphosphate and fructose diphosphate pool. Galactose would therefore be a gluconeogenic sugar in L. lactis subsp. cremoris FD1, but since fructose 1,6-diphosphatase is not present in this strain, galactose cannot serve as an essential biomass precursor (glucose-6P or fructose-6P) but only as an energy (ATP) source. Analysis of the growth energetics shows that transition from N limitation to limitation by glucose-6P or fructose-6P gives rise to a very high growth-related ATP consumption (152 mmol of ATP per g of biomass) compared with the value in cultures which are not limited by glucose-6P or fructose-6P (15 to 50 mmol of ATP per g of biomass). During lactose metabolism, the galactose flux through the tagatose-6P pathway (r(max) = 1.2 h) is lower than the glucose flux through glycolysis (r(max) = 1.5 h) and intracellular galactose-6P is dephosphorylated; this is followed by expulsion of galactose. Expulsion of a metabolizable sugar has not been reported previously, and the specific rate of galactose expulsion is up to 0.61 g of galactose g of biomass h depending on the lactose flux and the metabolic state of the bacteria. Galactose excreted during batch fermentation on lactose is reabsorbed and metabolized when lactose is depleted from the medium. In vitro incubation of galactose-6P (50 mM) and permeabilized cells (8 g/liter) gives a supernatant containing free galactose (50 mM) but no P(i) (less than 0.5 mM). No organic compound except the liberated galactose is present in sufficient concentration to bind the phosphate. Phosphate is quantitatively recovered in the supernatant as P(i) by hydrolysis with alkaline phosphatase (EC 3.1.3.1), whereas inorganic pyrophosphatase (EC 3.6.1.1) cannot hydrolyze the compound. The results indicate that the unknown phosphate-containing compound might be polyphosphate.     

GALT deficiency causes UDP-hexose deficit in human galactosemic cells

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.     

A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene

A new selection system based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene is presented. A broad range of plant species, including agronomically important crops such as maize and rice, is sensitive to low dosages of galactose. The toxicity of galactose is believed to be due to accumulation of galactose-1-phosphate, generated by endogenous galactokinase after uptake. Here, it is demonstrated that this toxicity can be sufficiently alleviated by the Agrobacterium tumefaciens-mediated introduction of the E. coli UDP-glucose:galactose-1-phosphate uridyltransferase (galT) gene, driven by a 35S-promoter, to allow transgenic shoots of potato and oil seed rape to regenerate on galactose containing selection media, resulting in high transformation frequencies (up to 35% for potato). Analysis of genomic DNA and UDP-glucose:galactose-1-phosphate uridyltransferase activity in randomly selected potato transformants confirmed the presence and active expression of the galT gene. The agricultural performance of transgenic potatoes was evaluated by monitoring the phenotype and tuber yield for two generations and these characters were found to be indistinguishable from non-transgenic controls. Thus, the galactose selection system provides a new alternative being distinct from conventional antibiotic and herbicide selection systems as well as so-called positive selection systems where the selective agent has a beneficial effect.     

Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

Lithium-mediated suppression of morphogenesis and growth in Candida albicans

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg(2+). In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway.     

Effects of 2-Deoxygalactose on Auxin-Induced Growth and Levels of UDP-Sugars in Higher Plants

Galactose inhibited the IAA-induced elongation of cells in segmentsof oat coleoptiles but not in segments of azuki bean and cucumberstems. In contrast, its analogs, 2-deoxygalactose and 2-deoxyglucose,inhibited the IAA-induced elongation of cells in these planttissues. In segments of cucumber stems, 2-deoxygalactose inhibitedformation of cell-wall polysaccharides with little effect onrespiration in terms of either reduced consumption of O₂ orlevels of ATP. 2-Deoxygalactose and 2-deoxyglucose caused arapid decrease in the levels of UTP and UDP-sugars with a concomitantincrease in levels of UDP in cucumber segments. These resultssuggest that 2-deoxysugars inhibit synthesis of cell-wall polysaccharidesby decreasing the levels of UDP-sugars, thereby inhibiting IAA-inducedelongation of cells in cucumber segments. 2-Deoxysugars can,therefore, be used as potential inhibitors with which to assessthe role of UDP-sugars in the IAA-induced elongation of cellsin dicotyledonous plants. (Received September 27, 1990; Accepted February 5, 1991)     

Restricted sugar uptake by sugar-induced internalization of the yeast lactose/galactose permease Lac12

Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.     

Enzymes of galactose utilization in the rat tapeworm, Hymenolepis diminuta.

1. Crude enzyme preparations from Hymenolepis diminuta contained galactokinase, galactose 1-phosphate uridyl transferase and UDPgalactose 4-epimerase activity, although their specific activities were low. 2. Galactose 1-phosphate non-competitively inhibited galactose phosphorylation. This inhibition, together with the low specific activities of the enzymes in the pathway of galactose utilization, probably accounts for the inadequacy of galactose as a main nutritive carbohydrate for development of the worm.     

Orotate decreases the inhibitory effect of ethanol on galactose elimination in the perfused rat liver.

1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose].     

Microdetermination of galactose and galactose 1-phosphate in dried blood spots

Newborn screening for galactosemia (galactose-1-phosphate uridyltransferase deficiency), as well as for other defects in galactose metabolism (galactokinase deficiency and uridine diphosphogalactose 4-epimerase deficiency), requires a method of determining both galactose and galactose 1-phosphate in dried blood. We have developed a sequential quantitative method for the microdetermination of galactose and galactose 1-phosphate that can be applied to 3-mm-diameter disks of dried blood and that can be used with a Technion Autoanalyser II equipped with a fluorometer.Galactose is determined by the fluorescence of NADH following treatment with β-galactose dehydrogenase and with the consequent reduction of NAD. The complete system includes alkaline phosphatase for the hydrolysis of galactose 1-phosphate, so that the total amounts of a galactose and galactose 1-phosphate are determined. For the measurement of galactose alone, alkaline phosphate is omitted from the system. The difference in fluorescence between that from the complete system and that from the alkaline phosphatase-omitted system yields the concentration of galactose 1-phosphate.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

The antagonism of IAA-induced hydrogen ion extrusion and coleoptile growth by diclofop-methyl

Diclofop-methyl (DM) (ester) was readily absorbed by peeled and unpeeled coleoptiles of wheat, Triticum aestivum L. cv. Waldron, and oat, Avena sativa L. cv. Garry. Substantial absorption of diclofop (acid) occurred only in peeled coleoptiles of the two species. IAA-induced acidification in peeled coleoptiles of both species was inhibited by 100 μ M DM or diclofop (acid) during a 3 to 4 h period. There was no recovery of acidification after DM or diclofop inhibition in oat coleoptiles; however, acidification in wheat coleoptiles recovered from inhibition by DM but not from diclofop. The recovery from DM inhibition may be due to a reduction in the diclofop pool derived from DM by efflux and metabolism (detoxification) in peeled wheat coleoptiles. Diclofop was not detoxified in oat coleoptiles. IAA-induced elongation of unpeeled oat coleoptiles was inhibited totally by 100 μ M DM but not by 100 μ M diclofop after 3.3 h of treatment. Wheat coleoptile elongation was relatively unaffected by either DM or diclofop. Basal elongation (no IAA) of both wheat and oat coleoptiles was inhibited by DM and diclofop. The inhibition by DM appeared to be irreversible, whereas the inhibition by diclofop was overcome by the addition of 10 μ M IAA.     

Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Effect of Xyloglucan Nonasaccharide on Cell Elongation Induced by 2,4-Dichlorophenoxyacetic Acid and Indole-3-acetic Acid

Xyloglucan nonasaccharide (XG9) is recognized as an inhibitorof 2,4-D-induced long-term growth of segments of pea stems.In the presence of 10^–5 M 2,4-D, inhibition by 10^–9M XG9 of elongation of third internode segments of pea seedlingswas detected within 2 h after the start of incubation, in someexperiments. Analysis by double-reciprocal (Lineweaver-Burk)plots of elongation in the presence of various concentrationsof 2,4-D, with or without XG9, gave parallel lines, indicatingthat XG9 inhibited 2,4-D-induced elongation in an uncompetitivemanner. XG9 did not influence the 2,4-D-induced cell wall loosening.Thus, XG9 does not fulfill the proposed definition of an "antiauxin". XG9 at 10^–11 to 10^–6 M did not influence IAA-inducedelongation of segments from pea third internodes, azuki beanepicotyls, cucumber hypocotyls, or oat coleoptiles. Inhibitionof IAA-induced elongation by XG9 was not observed even whenthe segments from pea or azuki bean were abraded. Furthermore,fucosyl-lactose at 10^–11 to 10^–4 M did not affectthe IAA-induced elongation of segments of pea internodes orof azuki bean epicotyls. XG9 may be incapable of inhibitingthe IAA-induced cell elongation (especially in oat) or, alternatively,the endogenous levels of XG9 may be so high that exogenouslyapplied XG9 has no inhibitory effect on IAA-induced elongation. (Received February 28, 1991; Accepted May 25, 1991)     

Galactose-1-Phosphatase in Rat Brain

A prominent galactose-1-phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl-Sephacel, hydroxylapatite, and Sephacryl S-300 columns. The galactose-1-phosphatase was separated from alkaline phosphatase, and from two forms of glucose-1-phosphatase. The three columns gave a 10-fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose-1-phosphate was the best substrate for the purified enzyme, followed by glucose-1-phosphate, which was hydrolyzed 40% as rapidly as galactose-1-phosphate. Galactose-1-phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose-1-phosphate hydrolysis. Mg2+ was required for activity, and supported half-maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L-homoarginine, and L-phenylalanine stimulated enzyme activity. Galactose-1-phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.1S. These values were used to calculate a molecular weight of 50,200 and a frictional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose-1-phosphate concentrations in patients with galactosemia.