Pyrophosphate-glucose Phosphotransferase Research Articles

Influence of the surface potential on the michaelis constant of membrane-bound enzymes: Effect of membrane solubilization

Detergents used at sufficiently high concentrations solubilize biological membranes, setting free their integral hydrophobic proteins. During this process the proteins may undergo serious alterations (review [I]). Mild detergents, especially those of non-ionic character, interact less drastically and therefore are routinely used for isolation of membrane-bound enzymes [2]. Nevertheless, even in these cases catalytic properties of the enzymes may be changed due to several factors such as alterations of secondary and tertiary structures, depletion of essential lipids, and presence of detergent molecules associated with the enzyme. This paper points to the abolition of the membrane surface potential as a factor influencing the activity of membrane enzymes after solubilization. It is a continuation of our studies in which the effect of the surface potential on Michaelis constants of a series of membrane-bound enzymes has been demonstrated [3-51. substrate. Glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was assayed with phenazine methosulphate as the primary electron acceptor and 2,6-dicbloroindophenol as the secondary acceptor as in [3]. The activity of monoamine oxidase (EC 1.4.3.4) was determined as in [lo] with dopamine as substrate by measuring oxygen uptake in the presence of 1 mM KCN. Acetylcholinesterase (EC 3.1 .1.7) was determined by measuring liberation of thiocholine from acetylthiocholine [ 111. NADPHcytochrome c reductase (EC 1.6.2.4) was measured according to [ 121. Glucose&phosphatase.(EC 3 .1.3.9) and the pyrophosphate-glucose phosphotransferase activity of this enzyme were assayed as in [ 131.

Genetic evidence for the common identity of glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase

We demonstrate that glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase activities are deficient in livers of patients with type I glycogen storage disease. This provides strong genetic evidence that these enzymatic activities reside in a single protein or share a common polypeptide chain.

Studies on a pyrophosphatase and glucose-6-phosphatase from Aspergillus oryzae

An inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) isolated from Aspergillus oryzae is associated with a glucose-6-phosphatase (glucose 6-phosphate phosphohydrolase, EC 3.1.3.9). It is also associated with a pyrophosphate-glucose phosphotransferase. These enzyme activities are heat stable, have acidic pH optima, and require no metallic ions for activity. Studies, including the determination of kinetic parameters, indicate that these catalytic activities are due to a single enzyme. This enzyme has a mol. wt of about 60 800 as determined by gel filtration. The enzyme also has low levels of acid phosphatase activity. A. oryzae possesses a second inorganic pyrophosphatase which has a low pH optimum and could be separated from the other catalytic activities.

Synthesis of glucose 6-phosphate by a 1,3-diphosphoglycerate-glucose phosphotransferase activity of liver microsomes

1. 1. Glucose 6-phosphate can be synthesized directly from 1,3-diphosphoglycerate and glucose in a reaction catalyzed by a liver and kidney microsomal enzyme whose properties resemble those of glucose-6-phosphatase (EC 3.1.3.9) with its accompanying inorganic pyrophosphate-glucose phosphotransferase activity. 2. 2. Specifically labeled radioactive 1,3-[1- 32P]diphosphoglycerate has been prepared and used to show that it is exclusively the acid anhydride phosphate on C-1 of that compound that is transferred to glucose. 3. 3. The possibilities that, in the system used, the glucose 6-phosphate synthesis occurs indirectly from 1,3-diphosphoglycerate by way of 3-phosphoglycerate kinase-catalyzed ATP formation or by way of phosphoglucomutase phosphorylation have been eliminated. 4. 4. Enzymatically catalyzed hydrolysis of 1,3-diphosphoglycerate occurs at 30°C and pH 6.0 in addition to the appreciable spontaneously occurring hydrolysis of the compound. 5. 5. The enzyme activity, like that of glucose-6-phosphatase and related enzyme activities, is greatly increased by pretreatment of the microsomes at about pH 10 or by detergent pretreatment. 6. 6. In activated preparations the pH optimum of the phosphotransferase reaction was 5.2–5.3 and the K m = 1.4 mM for 1,3-diphosphoglycerate and 0.08 M for glucose. 7. 7. Under comparable conditions, 1,3-diphosphoglycerate was 30–50% as effective a donor as PP i in the synthesis of sugar phosphates. Other sugars and sugar alcohols, as well as glycerol can be enzymatically phosphorylated by 1,3-diphosphoglycerate, the relative extent of phosphorylation of each acceptor compound being approximately the same as is observed with PP i as phosphate donor. The enzyme was unable to catalyze the phosphorylation of creatine with either PP i or 1,3-diphosphoglycerate as donor. 8. 8. PP i glucose (glycerol) phosphotransferase was found to be unable to catalyze the introduction of a second phosphate group into glycerol 3-phosphate or glucose 1-phosphate.

Use of chloroform to terminate the enzyme reaction in the assay of inorganic pyrophosphate-glucose phosphotransferase in rat liver

A method is described for the assay of inorganic pyrophosphate-glucose phosphotransferase from rat liver. The enzyme reaction is terminated by emulsifying the reaction mixture with chloroform. After dilution and centrifugation the reaction product, glucose 6-phosphate, is recovered quantitatively in the aqueous phase and may be assayed enzymically without further treatment. The principle of the method may have a more general application in the assay of other enzymes, especially in cases in which heat- and acid-labile reactants or products are involved, or when the product of the reaction is subsequently assayed enzymically.

Comparative studies of the responses of rat liver microsomal glucose-6-phosphatase and inorganic pyrophosphate-glucose phosphotransferase to phospholipase C treatment and phospholipid supplementation

1. 1.|Comparative studies of the responses of Glc-6- P phosphohydrolase and PP i-glucose phosphotransferase activities of rat liver microsomal glucose-6-phosphatase (EC 3.1.3.9) to in vitro phospholipid modifications have been carried out. Various conditions for assay enzymic activities were employed, and results compared. 2. 2.|Based on activity assays performed at pH 6 in the absence of added detergent, both activities decreased progressively and in parallel fashion with increasing length of exposure of microsomes to the action of phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). These activities of such treated preparation were increased by the addition of micellular dispersions of supplemental phospholipid (rat liver microsomal phospholipid preparations or, in one study, various classes of purified phospholipids). These observations provide yet another piece of evidence in support of catalysis of these two enzymic activities by a single microsomal enzyme. 3. 3.|The degree of restoration of activities towards original control levels through phospholipid supplementation following phospholipase C exposure of microsomes was much less when based on activity values obtained in the presence of the detergent deoxycholate than in its absence. 4. 4.|Enzymic activities of phospholipid-supplemented, phospholipase C-treated preparations were quite insensitive to stimulation by deoxycholate, while those of unexposed preparations were highly stimulated by this same detergent. 5. 5.|The pH-activity profile for PP i-glucose phosphotransferase activity obtained after phospholipase C exposure and phospholipid supplementation resembled strongly that noted with detergent-activated, otherwise untreated microsomal preparations, displaying a maximum at pH 5.6 in comparison with that at pH 4.5 exhibited by untreated microsomal preparations. 6. 6.|These observations are interpreted to indicate that phospholipid supplementation following phospholipase C exposure of rat liver microsomes does not lead to the restoration of microsomal glucose-6-phosphatase in its original form. Further, it is concluded that, in view of these findings and other observations in the literature, a reconsideration of the whole subject of the involvement of phospholipids in glucose-6-phosphatase action appears to be in order.

Inhibition of liver microsomal pyrophosphatase by free fatty acids

Treatment of the microsomal fraction of bovine liver with pancreatic lipase produced a time-dependent inactivation of acid pyrophosphatase activity. In the presence of bovine serum albumin, the activity of pyrophosphatase was protected during lipase treatment. Microsomal pyrophosphatase and related activities, glucose 6-phosphatase and pyrophosphate-glucose phosphotransferase were strongly inhibited in a parallel manner by inclusion of oleic acid in the assay mixture. The ability of various fatty acids to inhibit pyrophosphatase activity was dependent upon the length and unsaturation of the apolar chain. The results indicate that the free fatty acids released by lipase treatment are responsible for the observed inhibition and that bovine serum albumin protects the enzyme from inactivation by binding inhibitory free fatty acids.

Activation of microsomal glucose-6-phosphatase and associated pyrophosphatase and pyrophosphate-glucose phosphotransferase activities of human liver by aluminum oxide

Glucose-6-phosphatase activity and associated inorganic pyrophosphatase and pyrophosphate-glucose phosphotransferase activities of human liver microsomes was increased more than two-fold when the pH of the microsomal suspension was adjusted to 9.5 by Al 2O 3 powder and stirred overnight. Nearly 68% of glucose-6-phosphatase activity did not sediment at 105,000 × g after this procedure. The lipid content, pH optimum, Km, and antigenic characteristics of Al 2O 3 treated and intact microsomes were similar, but the enzymatic activity of treated fractions was more stable to heat (30°) and the RNA content was reduced by 50%. Electron microscopy indicated that the treated microsomes were essentially free of ribosomes and fragmented. The treatment appears to have dissociated the microsomal membrane by removing ribosomes, thus exposing more enzymatic active sites.

Different properties of glucose-6-phosphatase and related enzymes in rough and smooth endoplasmic reticular membranes

1. 1. Evidence presented supports the hypothesis that the endoplasmic reticular membrane enzyme, glucose-6-phosphatase, with its related activities, inorganic pyrophosphatase and inorganic pyrophosphate-glucose phosphotransferase, exists within the membrane in two different forms or different degrees of accessibility to substrate. In membrane areas having attached ribosomes the enzyme is predominately in an “activated” configuration while that in the smooth membranes is in a less active or potential form. 2. 2. The three enzyme activities studied have higher pH optima and, under normal assay conditions, greater specific activities in the ribosome-rich, “rough” subfractions of liver microsomes than in “smooth” subfractions of the same preparations. 3. 3. On activation by pretreatment with NH 4OH or deoxycholate, the enzymatic activities and pH optima of the smooth subfractions are shifted to higher values to a greater extent than are those of the rough. 4. 4. When comparisons are made on optimally activated samples, total, rough and smooth membranes exhibit approximately the same enzymatic activities and identical elevated pH optima. 5. 5. Despite great quantitative differences in the enzymatic activities of preparations from livers of fed, fasted, phenobarbital-treated and alloxan-diabetic rats, the same patterns of differences between enzyme activities of rough and smooth membranes was observed for all animals. 6. 6. Phenobarbital-treated animals, in which there is a large proliferation of smooth membranes, have been found by others to exhibit a lower specific activity of liver glucose-6-phosphatase than do control animals. This may be explained in part by the preponderance of less active enzyme in the smooth membranes.

Adenosine 5'-Triphosphate-Glucose and Phosphoenolpyruvate-Glucose Phosphotransferase Activities of Liver Microsomal Glucose 6-Phosphatase

Abstract Adenosine 5'-triphosphate-glucose and phosphoenolpyruvate-glucose phosphotransferase activities of rat liver microsomes have been studied. Evidence for the common identity of these activities with classical d-glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) is presented. All activities were equally susceptible to mild heating. Both phosphate compounds inhibited the hydrolysis of glucose 6-phosphate competitively, and Ki values thus assessed agreed closely with Km values for these relatively abundant high energy phosphate compounds functioning as phosphoryl donors in their respective synthetic reactions. The pH optima for both phosphotransferase activities, approximately 4.5 in the absence of detergent, were significantly shifted towards neutrality by the cationic detergent cetyltrimethylammonium bromide (cetrimide) and by palmityl coenzyme A. Kinetic studies at pH 5.5 revealed that Km (and Ki) values for phosphate substrates (but not glucose) were quite markedly altered by variations in ionic strength or cetrimide concentrations. In contrast, Vmax values were little affected. Responses of levels of ATP-glucose phosphotransferase activity to experimental diabetes, insulin or cortisone therapy, adrenalectomy, and acute fasting were studied with fresh microsomal suspensions and with such preparations to which deoxycholate had been added. The patterns of response observed were in all instances fully comparable with those previously noted with inorganic pyrophosphate-glucose phosphotransferase activity. Relative levels of the subject activities were compared with other hydrolytic and synthetic activities of the enzyme. It is concluded that physiologically significant roles for these activities are feasible, but because of the comparatively low levels of activities with ATP and phosphopyruvate and their acid pH optima, such roles, if manifest, are likely to be highly specialized.

Liver microsomal inorganic pyrophosphate-glucose phosphotransferase and glucose-6-phosphatase. Effects of diabetes and insulin administration on kinetic parameters

1. 1. Alterations in kinetic properties of rat hepatic glucose-6-phosphatase (( D-Gluc-6-P phosphohydrolase, EC 3.1.3.9) and associated PP i-glucose phosphotransferase resulting from alloxan diabetes and insulin treatment of such diabetic animals have been studied. 2. 2. Maximal reaction velocity values extrapolated to infinite substrate concentrations ( υ max ) and Michaelis constant ( K m ) values for all substrates were assayed with liver homogenates prepared from eight untreated normal, seven alloxan-diabetic, and seven insulin-treated diabetic animals. Average enzymic activity levels (units/mg liver protein) for the various groups of animals were calculated on the basis of υ max values. 3. 3. All assays were carried out both in the absence of added detergent and with homogenates which had been supplemented with sodium deoxycholate to 0.20% (w/v). 4. 4. As determined either in the presence or absence of detergent, K m values for glucose were not affected significantly by hormonal manipulations. 5. 5. K m values of both Gluc-6-P and PP i increased significantly in diabetes, based on assays carried out in the absence of detergent. These elevations were completely reversed by administration of insulin to diabetic animals, and were not apparent when homogenates prepared from livers of all three groups of animals were supplemented with deoxycholate prior to assay. 6. 6. Most significantly, preliminary treatment of homogenates with the detergent markedly potentiated responses of activity levels, based on extrapolated υ max values, to diabetes. The ratio of Gluc-6-P phosphohydrolase activity level in diabetic animals to activity level in normal animals increased from a value of 1.5, noted in the absence of detergent, to 3.5 based on assays with deoxycholate-supplemented homogenates. Even more dramatic potentiating effects of detergents on the diabetes response were noted with PP i-glucose phosphotransferase. With this activity, the ratio of υ max based activity levels in diabetic animals to ν max -based activity levels in normal animals increased from a statistically insignificant value of 0.90 in the absence of detergent to 4.2 when assessed in the presence of deoxycholate. Administration of insulin to diabetic rats reversed this response of ν max -based activity level values. These observations confirm our previous findings on the potentiating effect of detergent on responses of enzymic activity levels to experimental diabetes, and further, reveal that this effect persists even with substrate levels extrapolated to infinity and hence is not explainable simply on the basis of alterations in affinity of enzyme for substrates under the various experimental conditions. 7. 7. These patterns of effects of diabetes on kinetic parameters of the enzyme, as assessed both in the absence and presence of detergent supplementation of homogenates, closely resemble those previously noted in acutely fasted animals and contrast sharply with glucocorticoid-induced alterations.

Effects of cetyltrimethylammonium bromide on catalytic properties of kidney microsomal glucose-6-phosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphatase activities

The modifying effects of the cationic detergent cetyltrimethylammonium bromide (CTAB) on catalytic properties of kidney microsomal d- glucose-6-P phosphohydrolase (EC 3.1.3.9) and associated inorganic pyrophosphatase and PP 1-glucose phosphotransferase activities have been studied. Initial reaction velocities, apparent Michaelis constant values and apparent maximal reaction velocity values were determined at various H + concentrations with microsomal preparations which had been supplemented with the detergent to a final concentration (w/v), of 0.00, 0.05, 0.10, 0.20 or 0.30% 15 min prior to assay. Measurements of initial reaction velocities at various assay pH values indicated that CTAB treatment produced a shift in pH optimum of glucose-6-P phosphohydrolase activity towards acid pH values and in that of inorganic pyrophosphatase and phosphotransferase activities in the direction of neutrality. Both on the basis of initial reaction velocity values and apparent maximal reaction velocity values extrapolated to infinite substrate concentrations, lower concentrations of CTAB (0.05 and 0.10%, w/v) were found to elevate all activities, while higher concentrations (0.20 and 0.30%, w/v) were less effective in this respect or actually inhibited. In contrast with these polyphasic effects of the detergent on velocity values, apparent Michaelis constant values for all phosphate substrates, assayed at pH 4.5, 5.5, and 6.5, all decreased progressively as CTAB concentrations were increased from 0.05% to 0.30% (w/v). Plots (see J. S. Friedenwald and G. D. Meangwyn-Davies, in W. D. McElroy and B. Glass, The Mechanism of Enzyme Action, Johns Hopkins Press, Baltimore 1954, p. 180), of reciprocals of apparent Michaelis constant values for these compounds versus CTAB concentrations were linear, supporting the concept that CTAB acts as a “coupling” activator at lower concentrations and as a noncompetitive inhibitor at higher concentrations. Apparent Michaelis constants for PP i—0.3–0.4 mM with 0.30% (w/v) CTAB (pH 5.5)—are the lowest such values yet reported. The apparent Michaelis constant value for glucose in the phosphotransferase reaction, in contrast, was only modestly changed by CTAB treatment of microsomes. Several alternative mechanistic interpretations are considered briefly.

Activation in vitro of glucose-6-phosphatase, inorganic pyrophosphate glucose phosphotransferase and related enzymes. Relationship to microsomal membrane structure

1. 1. The elevation in enzymatic activity of certain microsomal enzymes caused in vitro by treatment with NH 4OH or deoxycholate has been studied in relationship to the appearance of the membranes in corresponding electron micrographs. 2. 2. When freshly prepared liver microscomes were treated at 0° with NH 4OH (about pH 9.8), activation of glucose-6-phosphatase (EC 3.1.3.9), inorganic pyrophosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphate glycerol phosphotransferase was not instantaneous. 3. 3. Exposure to NH 4OH for 15 to 30 min at 30° or contact for many hours at o° resulted in a maximum in vitro elevation of the enzyme activities that was approximately quantitatively equal to that observed instantaneously with such detergents as deoxycholate or Triton X-100. 4. 4. An electron microscopic study of NH 4OH-activated microsomes at 0° showed that ribosomes were largely retained on the membranes when about 50% of maximum stimulation of enzymatic activities occurred. Longer exposure at 0° or brief warming at 30° with NH 4OH, effecting full enzymatic activation, resulted in detachment of most of the ribosomes. The resulting membranes resembled those seen after exposure to inorganic pyrophosphate. 5. 5. The enzymatic activity of smooth microsomal membrane subfractions was capable of a percentage increase in vitro as great or greater than that of ribosome-rich rough membranes. 6. 6. Activation in vitro of these membrane enzymes is probably due to conformational changes in the membrane itself rather than to removal of ribosomes from their membrane attachment.

The Inhibition by Physiological Orthophosphate Concentrations of Hydrolytic and Synthetic Activities of Liver Glucose 6-Phosphatase

Abstract The inhibition by orthophosphate of glucose 6-phosphate phosphohydrolase and inorganic pyrophosphate-glucose phosphotransferase activities of rat liver glucose 6-phosphatase (EC 3.1.3.9) has been studied in detail. Significant inhibitions of both activities were noted over the entire range of pH values studied—pH 5.0 to 7.5 (hydrolase) or pH 5.0 to 6.5 (phosphotransferase). The detergents cetyltrimethylammonium bromide (Cetrimide) and lysolecithin significantly lowered Ki values for Pi with both activities. For example, at pH 7.5, a Ki value of 18 mm was noted in the absence of detergents, while preliminary treatment of microsomes with Cetrimide, to 0.05% (w/v), reduced this value to 2.0 mm, and inclusion of 0.4 mm lysolecithin in assay mixtures led to a decrease in Ki to 4.6 mm. Because of differences in the extents of accompanying modifying effects of detergents on Km values for substrates, net inhibition by Pi of glucose 6-phosphate phosphohydrolase activity was considerably more extensively potentiated by detergents than was that of phosphotransferase activity. Both in the absence and presence of detergents, inhibitions were kinetically competitive with respect to glucose 6-phosphate and pyrophosphate and non-competitive with respect to glucose in the phosphotransferase reaction. The mutually competitive nature of interactions of orthophosphate with adenosine triphosphate, bicarbonate, and pyrophosphate noted in inhibition studies carried out with phosphohydrolase activity also was shown. A physiologically significant regulatory role for Pi, through its effects on activities of this enzyme, is suggested on the basis of these experimental observations.

Rabbit intestinal microsomal inorganic pyrophosphate-glucose phosphotransferase: Sugar inhibitor specificity

Rabbit intestinal microsomal inorganic pyrophosphate-glucose phosphotransferase: Sugar inhibitor specificity

Inhibitory Effect of Physiological Bicarbonate Ion Levels on the Activities of Glucose 6-Phosphate Phosphohydrolase

Abstract Rat liver microsomal glucose 6-phosphate phosphohydrolase has been shown to possess two further catalytic activities: the hydrolysis of inorganic pyrophosphate to orthophosphate and a pyrophosphate-glucose phosphotransferase activity. Physiological concentrations of bicarbonate ion (10 to 50 mm) have been found to inhibit all three activities. A detailed kinetic analysis of the nature and extent of this inhibition has been carried out, with both fresh and deoxycholate-treated microsomes. The effect of pH on the degree of inhibition, at constant bicarbonate ion levels, suggests that bicarbonate ion rather than carbon dioxide is the inhibitory species. The inhibition by bicarbonate ion has been found to be competitive with respect to phosphate substrate and noncompetitive with respect to glucose. Measurements at varied substrate levels, when plotted according to the method of Dixon, show that 1/v is a linear function of [I], suggesting that bicarbonate ion is binding at the catalytic active site. The Ki values calculated for the three activities (≅20 mm) show close agreement, providing further support for the belief that a common active site, and possibly a common enzyme-phosphoryl intermediate, is involved in all three activities. It is suggested that the observed inhibition of glucose 6-phosphate phosphohydrolase by bicarbonate ion may explain the observation of Hastings and Longmore that increased bicarbonate-carbon dioxide levels result in increased glycogen accumulation in rat liver slices.

Human liver glucose-6-phosphatase characteristics of the detergent-solubilized enzyme

Detergent-solubilized human liver glucose-6-phosphatase ( d-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) has been found to have inorganic phyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) and pyrophosphate-glucose phosphotransferase activity (not yet classified). It is composed primarily of lipid and protein, with a small amount of RNA, and trace amounts of calcium and magnesium. Pigs and rabbits were immunized with it, and the antiserum also reacted with similar preparations from human kidney and intestine, and also from monkey and guinea pig liver. Liver from a child with glycogen storage disease, type I, did not produce a precipitin line in agar when it was diffused into the antiserum.

Liver microsomal inorganic pyrophosphate-glucose phosphotransferase: Alterations in activity-pH profiles by cortisone invivo

Liver microsomal inorganic pyrophosphate-glucose phosphotransferase: Alterations in activity-pH profiles by cortisone invivo

Biological Regulation of Liver Microsomal Inorganic Pyrophosphate-Glucose Phosphotransferase, Glucose 6-Phosphatase, and Inorganic Pyrophosphatase: Differential Effects of Fasting on Synthetic and Hydrolytic Activities

The effects of fasting on hydrolytic and synthetic activities of liver microsomal glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) have been investigated. Differential responses of the two types of activity were observed. Glucose 6-phosphate and inorganic pyrophosphate phosphohydrolase activity levels were elevated after a 48-hour fast, while inorganic pyrophosphate-glucose phosphotransferase activity responded relatively slightly, based on assays carried out in the absence of detergent. However, when homogenates were treated with deoxycholate prior to assay, parallel increases in all three activities were obtained. This latter observation constitutes additional substantiation for the multifunctional nature of microsomal glucose 6-phosphatase. The differential response patterns, and modifying effects of detergent thereon, were observed regardless of the hydrogen ion concentration in assay mixtures in the pH range 4.5 to 7. Kinetic analyses revealed that the differential response patterns were not due simply to fasting-induced changes in Km values, for the noted differences persisted when activity levels were expressed on the basis of Vmax values extrapolated to infinite concentrations of all substrates. Phosphotransferase activity of enzyme newly synthesized or newly activated in response to fasting appears to be blocked at the level of phosphoryl transfer from phosphoryl-enzyme intermediate to glucose. The observed differential patterns of response are consistent with the established hydrolytic role for the enzyme in fasted animals, and contrast with previously observed modes of response of these activities to glucocorticoid administration and experimental diabetes.

Regulation of liver microsomal inorganic pyrophosphate-glucose phosphotransferase, glucose-6-phosphatase and inorganic pyrophosphatase

Levels of liver inorganic pyrophosphate-glucose phosphotransferase, acid inorganic pyrophosphatase, and glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) have been assayed in normal, alloxan-diabetic, adrenalectomized and cortisone-treated adrenalectomized rats. Assays were carried out in the absence of added detergent, and with homogenates which had been supplemented with sodium deoxycholate or Triton X-100 to final concentrations of 0.004%, 0.012%, 0.04%, 0.20%, and 0.40%, w/v (deoxycholate) or v/v (Triton). Increases in levels of all activities were observed in the absence of detergent in cortisone-treated adrenalectomized rats compared with untreated adrenalectomized animals, and in diabetic compared with normal rats. Both detergents activated all three activities in all groups of animals. However, basic differences in the modes of response of these activities to cortisone administration as contrasted with insulin deprivation were apparent when assays were conducted with detergent-supplemented homogenates. As detergent concentration was increased, a progressive diminution was noted in the ratios of activity in cortisone-treated compared with untreated adrenalectomized animals. Statistically significant differences in activity level values for the two groups of animals disappeared when deoxycholate concentrations were 0.20% or 0.40%; Triton X-100 also effected a marked diminution in activity level ratios under these conditions. In contrast, differences between comparable activity level values for diabetic compared with normal animals were progressively magnified with increasing concentration of both detergents. These observations indicate that the detergent effects involved are general in nature, and point up the complexity of mechanisms involved in responses of activities of this enzyme to hormonal manipulations. They also raise a question as to the conditions which properly should be employed to assess also raise a question as to the conditions which properly should be employed to assess alterations in functional levels, in vivo, of these activities in future horm and nutritional studies.