Phosphoglucoisomerase Activity Research Articles

Phosphoglucoisomerase Is an Important Regulatory Enzyme in Partitioning Carbon out of the Calvin-Benson Cycle.

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI Km for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the Km of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.

Normal activities of AMP-deaminase and adenylate kinase in patients with McArdle disease

During physical activity in McArdle patients, little or no lactate is released in the skeletal muscle. However, excessive ammonia production has frequently been reported in these patients. Production of ammonia is catalysed by AMP deaminase (AMPD) and adenylate kinase (AK). The activities of AMPD and AK along with housekeeping enzyme phosphoglucoisomerase (PGI) were measured in 11 genetically confirmed McArdle patients and compared with 27 healthy controls. The AMPD and AK activities were not significantly different in patients and controls. The activity of PGI was significantly higher in patients than in controls suggesting compensation of the impaired glycogenolysis in McArdle. The ratios of activities of AMPD and AK over PGI were significantly lower in patients than in controls. High ammonia production in McArdle patients is not based on enzyme induction of AMPD and AK but possibly due to kinetic activation of the enzyme AMPD by increased concentration of the substrate AMP.

Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50% while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64% respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85% increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose.

Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis.

HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight.

Phosphotransferase System-Mediated Glucose Uptake Is Repressed in Phosphoglucoisomerase-Deficient Corynebacterium glutamicum Strains

Corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum Δpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum Δpgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized l-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmid-encoded ptsG. l-Lysine yields and productivity with C. glutamicum Δpgi(pBB1-ptsG) were significantly higher than those with C. glutamicum Δpgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTS-mediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast l-lysine production.

Use of flux pre‐analysis to enable 13C tracer studies in pyruvate kinase‐deficient Escherichia coli

Pyruvate kinase-deficient Escherichia coli (PB25) is a low by-product-producing yet fast-growing mutant that has been shown to have technological potential. Determining the flux limits through finding the extreme point flux sets was previously reported to identify alternate metabolite trafficking scenarios. Previously, the extreme point flux sets were used to design tracer experiments; however, variation in extracellular measurements was not considered, and reaction reversibility was assumed to be low to moderate. In this study, we examined the utility of limiting the fluxes and predetermining the trafficking scenarios in PB25, including confirmation of quasi-linearity between extreme points to ensure sensitivity is maintained. The effects of variation in extracellular measurements and reaction reversibilities were also examined. Tightened flux limits reduced the nonlinearity between label distribution and fluxes. For low to moderate reversibility, contrast was also preserved. However, for highly reversible phosphoglucoisomerase activity, information from common analytes could lead to a flux solution that is biased towards one extreme point. Based on the PB25 model, some suggestions are provided for how predetermining flux limits and trafficking scenarios could enable flux identification in larger network problems.

Effect of tellurite-mediated oxidative stress on the Escherichia coli glycolytic pathway

To unveil the metabolic impact of tellurite in the bacterial cell, the effect of this toxicant on the expression and activity of key enzymes of the Escherichia coli glycolytic pathway was analyzed. E. coli exposure to tellurite results in: (i) increased glucose consumption, which was paralleled by an increased expression of the glucose transporter-encoding gene ptsG, (ii) augmented phosphoglucoisomerase activity and pgi transcription, (iii) decreased activity of the enzymatic regulators phosphofructokinase and pyruvate kinase. In spite of these observations, increased intracellular pyruvate, phosphoenol pyruvate and phosphorylated sugars was observed. E. coli lacking key glycolytic enzymes was considerably more sensitive to tellurite than the parental, isogenic, wild type strain. Taken together, these results suggest that increasing the availability of key metabolites (pyruvate, phosphoenol pyruvate, NADPH), required to respond to tellurite mediated-stress, E. coli shifts the carbon flux towards the pentose phosphate pathway thus facilitating the functioning of the Entner-Doudoroff pathway and/or the glycolytic productive phase.

Phosphoketolase Pathway Dominates in Lactobacillus reuteri ATCC 55730 Containing Dual Pathways for Glycolysis

Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro 31P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.

Cadmium induced changes in carbohydrate status and enzymes of carbohydrate metabolism, glycolysis and pentose phosphate pathway in pea

With 50 μmol/l CdCl 2, reduction of about 40% in shoot and 70% in root lengths was observed in pea seedlings. The reduction in seedling growth was accompanied by decreased water content of shoots and roots. Increasing trend of sucrose was observed in shoots of cadmium-stressed seedlings. Cotyledons of stressed seedlings had lower α-and β-amylase activities resulting in their higher starch content. Reducing sugar and sucrose contents were not significantly affected in roots of cadmium-stressed seedlings but starch content was low in comparison with normal seedlings which appeared to be due to lesser conversion of sucrose into starch rather than its rapid hydrolysis. In general activities of acid and alkaline invertases were low in stressed seedlings with exception to cotyledons where alkaline invertase was more under stress. Cadmium-stressed seedlings had lower sucrose synthase and sucrose phosphate synthase (SPS) activities in roots and shoots but cotyledons had 10–15% higher SPS activity. Phospho glucoisomerase activity was maximum in cotyledons showing its involvement in conversion of glucose-1-phosphate to glucose-6-phosphate whereas the activity of this enzyme was almost negligible in roots. Exogenous cadmium up regulated the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in cotyledons whereas activities of these enzymes were down regulated by 40–50% in shoots. Higher activities of hexokinase (10–15%) and phospho glucoisomerase in roots and shoots of stressed seedlings indicated that hexoses are preferably channeled towards glycolysis than pentose phosphate pathway under cadmium toxicity.

Antidiabetic effect ofT. arjuna bark extract in alloxan induced diabetic rats

The present study was carried out to evaluate the antidiabetic effect of T. arjuna stembark extract and to study the activities of hexokinase, aldolase and phosphoglucoisomerase, and gluconeogenic enzymes such as glucose-6-phosphatase and fructose-1,6-diphosphatase in liver and kidney of normal and alloxan induced diabetic rats. Oral administration of ethanolic extract of bark (250 and 500mg/kg body weight) for 30 days, resulted in significant decrease of blood glucose from 302.67±22.35 to 82.50±04.72 and in a decrease in the activities of glucose-6-phosphatase, fructose-1,6-disphosphatase, aldolase and an increase in the activity of phosphoglucoisomerase and hexokinase in tissues. However, in the case of 250 mg/kg body weight of extract, less activity was observed. The study clearly shows that the bark extract ofT. arjuna possesses potent antidiabetic activity.

Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess.

The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate. Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)). No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K. lactis and other known Crabtree-negative yeasts. During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions. When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change. Our results suggest that the low tendency for ethanol formation in K. marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel.

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase), and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI) and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH) and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR.

The reversible conversion between D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-[1H] EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates. The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-[2-13C]glucose 6-phosphate and beta-D-[2-13C]fructose 6-phosphate. Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates. The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme. The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D-fructose 6-phosphate both as a substrate and a product, contrary to previous proposals. This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase.

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates

When D-[2-3H]glucose 6-phosphate mixed with the unlabeled ester is converted to D-[1-3H]fructose 6-phosphate and 3HOH in the phosphoglucoisomerase reaction and then to D-[1-3H]fructose 1,6-bisphosphate in the phosphofructokinase reaction, the specific radioactivity of the latter metabolite and the production of 3HOH relative to the total generation of tritiated end products are both inversely related to the concentration of phosphofructokinase. In human erythrocytes, the modeling of D-[2-3H]glucose metabolism, based on the activity of phosphoglucoisomerase in cell homogenates and on the steady-state content of D-glucose 6-phosphate and D-fructose 6-phosphate in intact cells, indicates that the back-and-forth interconversion of these esters is about five-times higher than the net glycolytic flux. Yet, the production of 3HOH from D-[2-3H]glucose is about 20% lower than the net glycolytic flux, as judged from the production of 3HOH from D-[5-3H]glucose. Thus, an incomplete detriation of D-[2-3H]glucose is not incompatible with an extensive interconversion of hexose 6-phosphates in the reaction catalyzed by phosphoglucoisomerase.

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.

Gene silencing in a polyploid homosporous fern: paleopolyploidy revisited.

Because of their high chromosome numbers, homosporous vascular plants were considered paleopolyploids until recent enzyme electrophoretic studies rejected this hypothesis by showing that they express only diploid numbers of isozymes. In polyploid sporophytes of the homosporous fern pelleae rufa, however, progressive diminution of phosphoglucoisomerase activities encoded by one ancestral genome culminates in tetraploid plants exhibiting a completely diploidized electrophoretic phenotype for this enzyme. The demonstration that such gene silencing can make a polyploid fern look isozymically like a diploid questions the validity of isozyme evidence for testing the paleopolyploid hypothesis and supports the proposed role of polyploidization followed by genetic diploidizaton in the evolutionary history of homosporous pteridohytes.

Metabolic Characteristics Associated with Starch, Protein, and Oil Deposition in Developing Maize Kernels

The physiological processes that control relative starch, protein, and oil concentration of maize(Zea mays L.) kernels are not well defined. In this study, associationS between Metabolic Characteristics of developing kernels and specific storage product accumulation in mature kernels were identified. In a field experiment, kernels of maize genotypes with widely divergent chemical composition were harvested 50 days post pollination (DPP) to compare starch, protein, and oil concentrations with enzyme activities, sugar,and aminoc acid levels of kernels harvested 20 DPP. Mature kernels starch concentrations were correlated with immature kernel sucrose concentrations and ADP‐glucose pyrophosphorylase activities. Amino acid concentrations in immature kernels were correlated with the activities of phosphoglucoisomerase, Phosphoglucomutase, pyrophosphate: fructose 6‐ phosphalt 1‐phosphotransferase, and aldolasea and with mature kernel protein concentrations, but negatively correlated with starch concentrations. Oil concentrations were positively correlated with mature embryo dry weights and with malate dehydrogenase activities in isolated embryos harvested 20 DPP. The results suggest a complex interaction of sugars and amino acids supplied to the kernel by the plant affecting enzyme activities and storage product accumulation the developing kernel. High‐starch genotypes supplied more sucrose to their kernels, had higher ADP‐glucose pyrophosphorylase activity, and greater starch accumulation. High‐protein genotypes supplied more amino acids to their kernels, and had enhanced expression of a number of enzymes that may contribute to the greater protein accumulation. These data suggest that nutrient supply interacts with expression of enzyme activities in developing kernels to determine starch and protein concentrations in mature kernels.

Hexose metabolism in pancreatic islets: Enzyme-to-enzyme tunnelling of hexose 6-phosphates

The fate of unlabelled d-glucose and d-[2- 3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of d-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between d-[2- 3H]glucose 6-phosphate and d-[1- 3H] fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from d-[2- 3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from d-[2- 3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of d-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A) + RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an M r of 61310. The nucleotide sequence of the gene as well as the 5′ and 3′ flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.

Mutants of the obligate methylothroph Methylobacillus flagellatum KT defective in genes of the ribulose monophosphate cycle of formaldehyde fixation

An approach to the isolation of mutants deficient in key enzymes of C1 metabolism was worked out for the obligate methylotroph Methylobacillus flagellatum KT. The isolation of mutants in genes of the ribulose monophosphate (RMP) cycle of formaldehyde assimilation and oxidation is based on selection of temperature sensitive (ts) mutants followed by enzymatic assays. Standard methods of determination of activities of hexulose phosphate synthase/hexulose phosphate isomerase (HPS/HPI), phosphoglucoisomerase (PGI), glucose 6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (GND) were modified for screening the ts-mutants for the presence of RMP keyenzyme activities. Among the 500 ts-mutants investigated, nine mutants were defective in PGI activity and two in GDP activity. The defective enzymes were characterized by a high rate of inactivation at increased temperatures. At 50° C the proteins studied were completely inactivated during 2 h, whereas the native enzymes maintained more than 80% activity.