Présence des enzymes catalysant la biosynthèse d'UDP glucose dans une fraction légère de l'appareil de Golgi des hépatocytes de chat

A Golgi-rich fraction is prepared from cat hepatocytes by the means of a four-step sucrose density gradient. The material applied to this gradient is composed either of smooth microsomes prepared from healthy animals, or of total microsomes prepared from cat treated by 50 per cent ethanol (0.6 g/100 g body weight, administered by stomach tube).A light fraction (d : 1.10) is obtained by the two procedures. It does not show any glucose-6-phosphatase activity, but is enriched in sialyltransferase, known as a marker enzyme for Golgi apparatus. It also contains the three enzymes implicated in the biosynthetic pathway for UDP-glucose (glucokinase, phosphoglucomutase and UTP : glucose-1-phosphate uridylyltransferase).UDP-glucose being the ultimate substrate in membranous glucosylation reactions, these results could support the hypothesis that sugar-nucleotides necessary for the glycoprotein biosynthesis are produced in the Golgi vesicles directly.     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.     

A method for rapid isolation of rough and smooth microsomes and golgi apparatus from rat liver in the same sucrose gradient

A technique is presented for the rapid isolation, by centrifugation in the same sucrose gradient, of rough and smooth surfaced microsomes and a Golgi apparatus enriched fraction from rat liver. Ethanol treatment is not necessary. The purity and recovery of the isolated fractions, as judged from determinations of marker enzymes and morphological analyses, are similar to previous data obtained with more complicated procedures. The method is based on adding a 10000 g supernatant to a discontinuous sucrose gradient ranging from bottom to top (1.30 M–15 mM CsCl, 1.15 M–15 mM CsCl, 10000 g supernatant in 1.05 M–10 mM CsCl, 1.05 M sucrose and 0.3 M sucrose). The Golgi apparatus enriched fraction bands at the border line of 0.3/1.05 M sucrose. The smooth microsomes band at the bondary of 1.15/1.30 M sucrose and finally the rough microsomes are collected at the bottom of the gradient. All fractions are obtained within h.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain

Abstract: Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [³H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2', 3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34, 880 x g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties.     

Isolation and characterization of Golgi membranes from bovine liver

Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60–80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.     

Purification of a UTP: d-Glucose-1-phosphate uridylyl-transferase from Golgi apparatus of cat liver by affinity chromatography on UTP-agarose

UDP-glucose pyrophosphorylase from Golgi apparatus solubilized by detergent has been purified 100-fold from microsomes by affinity chromatography on UTP-agarose. The purified enzyme has apparent M_r 270,000 and isoelectric pH 3.9 against 360,000 and 4.2 for soluble enzyme. According to these characteristics, UDP-glucose pyrophosphorylase from Golgi apparatus is different from cytosolic enzyme.     

Phospholipid synthesis in a membrane fraction associated with mitochondria

A crude rat liver mitochondrial fraction that was capable of the rapid, linked synthesis of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) labeled from [3-3H] serine has been fractionated. PtdSer synthase, PtdEtn methyltransferase, and CDP-choline:diacylglycerol cholinephosphotransferase activities were present in the crude mitochondrial preparation but were absent from highly purified mitochondria and could be attributed to the presence of a membrane fraction, X. Thus, previous claims of the mitochondrial location of some of these enzymes might be explained by the presence of fraction X in the mitochondrial preparation. Fraction X had many similarities to microsomes except that it sedimented with mitochondria (at 10,000 x g). However, the specific activities of PtdSer synthase and glucose-6-phosphate phosphatase in fraction X were almost twice that of microsomes, and the specific activities of CTP:phosphocholine cytidylyltransferase and NADPH:cytochrome c reductase in fraction X were much lower than in microsomes. The marker enzymes for mitochondria, Golgi apparatus, plasma membrane, lysosomes, and peroxisomes all had low activities in fraction X. Polyacrylamide gel electrophoresis revealed distinct differences, as well as similarities, among the proteins of fraction X, microsomes, and rough and smooth endoplasmic reticulum. The combined mitochondria-fraction X membranes can synthesize PtdSer, PtdEtn, and PtdCho from serine. Thus, fraction X in combination with mitochondria might be responsible for the observed compartmentalization of a serine-labeled pool of phospholipids previously identified (Vance, J. E., and Vance, D. E. (1986) J. Biol. Chem. 261, 4486-4491) and might be involved in the transfer of lipids between the endoplasmic reticulum and mitochondria.     

Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.     

Properties of a rat liver smooth microsomal subfraction not aggregated by Mg2+

A smooth microsomal fraction (smooth II microsomes) was earlier isolated and characterized in a number of investigations. Using a three-layer discontinuous sucrose gradient containing Mg²⁺ this fraction was divided into two subfractions (IIa and IIb) by a single centrifugation. The smooth IIa fraction proved to be a purified smooth microsomal fraction of specific composition. It contains high amounts of cytochromes $\text{b}{}_5$ and P-450, low activities of other electron transport enzymes and glucose-6-phosphatase, and no UDP-glucuronic acid transferase. No membrane or enzyme synthesis is induced in this subfraction by treatment with phenobarbital or methylcholanthrene. It appears that the membranes of smooth IIa microsomes derive from the smooth endoplasmic reticulum and are devoted to specific functions.     

GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES : I. Isolation Procedure and Morphological Characterization

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ~90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ~13 x 10⁶ g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Multiple forms of glycosyltransferases in Golgi complex membrane fractions

The distribution of multiple forms of galactosyltransferase (EC 2.4.1.22) and sialyltransferase (EC 2.4.99.1) from the microsomes and Golgi complex membrane fractions of rat liver was investigated. Three fractions of Golgi membranes, namely GF1, GF2, and GF3, differing in their morphology and marker enzyme activity, were obtained. A simultaneous increase of glycosyltransferases under study was observed in fractions GF3 less than GF2 less than GF1. Using isoelectrofocusing, the presence of at least 6-8 forms of galactosyl- and sialyltransferases in the microsomes and Golgi fraction was revealed. The distribution patterns of multiple forms along the pH gradient for each membrane fraction were found to be identical. However, the ratios of highly active and low active forms were specific for each fraction. The similarity of multiple form spectra for galactosyl-and sialyltransferase suggest their tight functional interaction and a possible "en block" packing of membrane glycosyltransferases.     

Characterization of the <Emphasis Type="Italic"> ugpG </Emphasis>gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer <Emphasis Type="Italic"> Sphingomonas paucimobilis </Emphasis>ATCC 31461

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.     

Isolation and Partial Characterization of Plasmalemma from Quiescent Schwann Cells in Denervated Cat Sciatic Nerve

Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89% in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8-10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M:0.85 M and 0.85 M:1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5'-nucleotidase, 2',3'-cyclic nucleotide 3'-phosphohydrolase. and specific [3H]ouabain binding, 4.8-, 3.0-, and 5.7-fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatase (9, 13, and 15% of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Strain improvement and metabolic flux modeling of wild-type and mutant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. NX-3 for synthesis of exopolysaccharide welan gum

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N⁺ ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation.     

Isolation by affinity chromatography of specialized membrane fractions from cat liver microsomes]

Microsomal glucokinase is solubilized by incubation in the presence of several metabolites. After solubilization of the enzymes, the membranes present free sites for specific binding of glucokinase, therefore, they can be purified by affinity chromatography on Sepharose--ATP-glucokinase. This method yields membranous vesicles which contain, in addition to glucokinase, uridylyl-transferase, phosphoglucomutase, sialyl-transferase and adenylate cyclase. Galactosyl-transferase, glucose-6-phosphatase and NADPH cytochrome c reductase are absent. It appears that functionally related enzyme from UDP-glucose biosynthesis are aggregated onto specific patches of the membrane, most likely from Golgi apparatus.     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.