Individual Enzyme Activities Research Articles

Dynamic Disorder and Stepwise Deactivation in a Chymotrypsin Catalyzed Hydrolysis Reaction

In situ observation of the catalytic activity of individual α-chymotrypsin enzymes reveals a novel pathway for spontaneous deactivation. Rather than deactivating abruptly in a one-step process, the enzyme seems to struggle for life; the activity decreases stepwise with intermittent inactive periods before deactivating irreversibly. During the active periods, dynamic disorder and memory effects are observed, originating from conformational fluctuations within the enzyme's structure.

Focused microarray analysis of glyco‐gene expression in human glioblastomas

Altered glycosylation has been linked to cancer cell metastasis and invasivity. We have previously shown that expressing a specific sialyltransferase gene in gliomas inhibited tumor formation, in vivo. In order to identify other "glyco-gene" targets with therapeutic potential, focused 45-mer oligonucleotide microarrays were constructed containing all of the cloned human glyco-genes. Gene expression profiles of normal human brain and malignant gliomas were compared and microarray datasets analyzed using significance analysis of microarrays algorithms. There were 11 genes more highly expressed in gliomas compared to normal brain and 25 genes more highly expressed in normal brain compared to gliomas. Among the most noteworthy were high levels of MAN2A2 and ST6GalNAcV in normal brain tissue and high levels of POFUT1 and CHI3L1 in the gliomas, all changes corroborated by qRT-PCR. Historically, identification of changes in tumor-associated glycoconjugate expression was obtained by measuring individual enzyme activities or structural changes of specific molecules. With microarray technology, it is possible to measure all of genes associated with glycoconjugate biosynthesis and degradation simultaneously. Our data demonstrate that there are many significant and novel differences in glyco-gene expression that represent potential targets for the development of therapeutics for the treatment of brain tumors.

Improved analytical sensitivity reveals the occurrence of gender-related variability in diamine oxidase enzyme activity in healthy individuals

Objectives: Serum diamine oxidase (DAO; EC 1.4.3.6) activity is often employed as a clinical indicator of the integrity of intestinal mucosa. However, interindividual variation in enzyme activity and reference values for healthy women and men have not been studied. Design and methods: DAO activity was measured by using cadaverine coupled to O-dianisidine oxidation in 50 healthy individuals. Results: The mean activity was 7.59 ± 3.67 U/L for women and 2.38 ± 0.71 U/L for men ( p < 0.001). Conclusions: Gender is a major determinant for DAO activity in healthy subjects.

HO-1 Overexpression Attenuates Endotoxin Effects on CAT-2 Isozymes Expression

l-arginine transport mediated by type-2 cationic amino acid transporter (CAT-2) isozymes is one crucial mechanism that regulates nitric oxide (NO) production via inducible nitric oxide synthase (iNOS). We sought to investigate the effects of heme oxygenase-1 (HO-1) overexpression on CAT-2 isozymes, e.g., CAT-2, CAT-2A, and CAT-2B. Adult male Sprague Dawley rats were allocated to receive lipopolysaccharide (LPS), normal saline, hemin (a HO-1 inducer), tin protoporphyrin (SnPP, a HO-1 inhibitor), LPS plus hemin, or LPS plus hemin plus SnPP. After maintaining for 6 h, rats were sacrificed and the expression and activity of individual enzyme was evaluated. LPS increased HO activity, HO-1 concentration, NO production, l-arginine transport, and concentrations of iNOS, CAT-2, and CAT-2B in rat lungs and kidney. LPS also increased HO activity, HO-1 concentration, NO production, l-arginine transport, and iNOS concentration but decreased CAT-2 and CAT-2B concentrations in rat liver. LPS increased CAT-2A concentration in rat liver but did not affect CAT-2A concentration in rat lungs and kidney. Hemin further increased HO activity and induced HO-1 overexpression in the lungs, kidney, and liver from LPS-treated rats. In addition, the effects of LPS on NO production, l-arginine transport, and concentrations of iNOS and CAT-2 isozymes were significantly attenuated by hemin. SnPP, on the other hand, reversed the effects of hemin. HO-1 overexpression significantly attenuates endotoxin-induced increases in NO production and l-arginine transport. Induction of HO-1 overexpression also significantly attenuates the effects of endotoxin on the expression of iNOS and CAT-2 isozymes in septic rats.

Single-molecule studies of complex systems: the replisome

A complete, system-level understanding of biological processes requires comprehensive information on the kinetics and thermodynamics of the underlying biochemical reactions. A wide variety of structural, biochemical, and molecular biological techniques have led to a quantitative understanding of the molecular properties and mechanisms essential to the processes of life. Yet, the ensemble averaging inherent to these techniques limits us in understanding the dynamic behavior of the molecular participants. Recent advances in imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and record "molecular movies" that provide insight into their dynamics and reaction mechanisms. An important future goal is extending the applicability of single-molecule techniques to the study of larger, more complex multi-protein systems. In this review, the DNA replication machinery will be used as an example to illustrate recent progress in the development of various single-molecule techniques and its contribution to our understanding of the orchestration of multiple enzymatic processes in large biomolecular systems.

The anti-oxidative defence system in the isolated rat uterus during spontaneous rhythmic activity

Possible interactions between nitric oxide donors, reactive oxygen species and anti-oxidative defence enzymes led us to determine the activities of anti-oxidative defence enzymes in isolated uterine smooth muscle before and after spontaneous rhythmic activity ex vivo. For our experiments we used isolated uteri from female Wistar rats. Our results showed an increase in total superoxide dismutase (SOD) and Mn SOD activities in uterine smooth muscle after spontaneous contractions when compared with nonexercised uterine smooth muscle. The activity of catalase (CAT) and glutathione preoxidase (GSH-Px) were also increased. No statistically significant changes in the activities of glutathione reductase (GR) and CuZn SOD were found. It is known that an organism's anti-oxidative defence system (guarding against excessive reactive oxygen species generation) requires balanced increments in its individual anti-oxidative enzyme activities rather than increases in the activity of only some enzymes without increases in others. Thus, we may conclude that some adaptive responses are found in exercised uterine smooth muscle but are not complete. Therefore, our results indicate that changes in anti-oxidative enzyme activities may influence the results of the examination of substances ex vivo.

Enzymatic Activity in Extracts of Allergy-Causing Astigmatid Mites

Many of the previously characterized allergens of house dust mites are known to be proteases, and this enzymatic activity is thought to contribute to their allergenicity. Other astigmatid mites, including stored-product mites and the ectoparasitic itch mite, Sarcoptes scabiei De Geer, are also known to be allergenic, but little or nothing is known about their enzymatic activities. The purpose of this study was to characterize the enzymatic activities present in extracts of the parasitic itch mite and from eight other species of free-living astigmatid mites. Extracts were prepared from one parasitic mite (S. scabiei), five stored-product mites (Chortoglyphus arcuatus (Troupeau), Lepidoglyphus destructor (Schrank), Blomia tropicalis Bronswijk, Cock, Oshima, Tyrophagus putrescentiae (Schrank), and Acarus siro L.), and three house dust mites [Dermatophagoidesfarinae Hughes, Dermatophagoides pteronyssinus (Troussart), and Euroglyphus maynei (Cooreman) ]. ApiZym strips were used to screen for the presence of 19 individual enzyme activities. Digestion of nine other substrates was evaluated by spectrophotometric or electrophoretic methods. All mite extracts exhibited some form of phosphatase, esterase, aminopeptidase, and glycosidase activity, although their substrate specificities varied considerably. Itch mite extract did not possess detectable serine peptidase activity nor was it able to hydrolyze gelatin or casein, whereas all other mite extracts exhibited these activities. Storage mite extracts possessed enzymes capable of degrading the widest range of substrates, whereas itch mite extract had the most limited proteolytic capacity. Extracts of nine species of allergy-causing astigmatid mites contain wide and diverse repertoires of enzymatic activities. These catalytic activities may be important contributors to the induction and manifestation of inflammatory and immune responses to mites in patients.

Liver disease selectively modulates cytochrome P450–mediated metabolism

The liver plays a significant role in drug metabolism; thus it would be expected that liver disease may have a detrimental effect on the activity of cytochrome P450 (CYP) enzymes. The extent to which the presence and severity of liver disease affect the activity of different individual drug-metabolizing enzymes is still not well characterized. The purpose of this study was to assess the effect of liver disease on multiple CYP enzymes by use of a validated cocktail approach. The participants in this investigation were 20 patients with different etiologies and severity of liver disease and 20 age-, sex-, and weight-matched healthy volunteers. Liver disease severity was categorized by use of the Child-Pugh score. All participants received a cocktail of 4 oral drugs simultaneously, caffeine, mephenytoin, debrisoquin (INN, debrisoquine), and chlorzoxazone, as in vivo probes of the drug-metabolizing enzymes CYP1A2, CYP2C19, CYP2D6, and CYP2E1, respectively. The primary end points were measurements of specific CYP metabolism indexes for each enzyme. Mephenytoin metabolism was significantly decreased in both patients with mild liver disease (Child-Pugh score of 5/6) (-63% [95% confidence interval (CI), -86% to -40%]; P = .0003) and patients with moderate to severe liver disease (Child-Pugh score >6) (-80% [95% CI, -95% to -64%]; P = .0003). In comparison with control subjects, the caffeine metabolic ratio was 69% lower (95% CI, -85% to -54%; median, 0.14 versus 0.62; P = .0003), the debrisoquin recovery ratio was 71% lower (95% CI, -96% to -47%; median, 0.10 versus 0.65; P = .012), and the chlorzoxazone metabolic ratio was 60% lower (95% CI, -91% to -29%; median, 0.21 versus 0.83; P = .0111) in patients with moderate to severe liver disease. All 4 drugs showed significant negative relationships with the Child-Pugh score. CYP enzyme activity is differentially affected by the presence of liver disease. We propose that the data can be explained by the "sequential progressive model of hepatic dysfunction," whereby liver disease severity has a differential effect on the metabolic activity of specific CYP enzymes.

Myosin expression levels and enzyme activity in juvenile spotted wolffish (Anarhichas minor) muscle: a method for monitoring growth rates

The activity of glycolytic enzymes and the expression levels of myosin RNA was monitored in the white muscle of juvenile spotted wolffish (Anarhichas minor) reared under different temperature regimes. A group of individually tagged juvenile spotted wolffish was reared for 6 months at 4, 6, 8, and 12 °C. After the rearing trial, biopsy samples were taken from white muscle of each individual and the relationship between individual growth, enzyme activity, and myosin expression was investigated. A positive relationship between the activities of two glycolytic enzymes (pyruvate kinase and lactate dehydrogenase) and individual growth rate was observed. Using real-time polymerase chain reaction (PCR) and specially developed primers for myosin mRNA and 18S rRNA for spotted wolffish, we were able to detect differences in the relative myosin expression between experimental groups, and a positive relationship between myosin expression and specific growth rates was observed. These methods may be useful as an indicator of growth rate in wild fish and a fast and reliable indicator of growth potential under culture conditions. The method also has the potential to measure differences in white muscle synthesis in fish reared under variable environmental parameters and during different life history stages.

Interdomain Communication in DNA Topoisomerase II: DNA BINDING AND ENZYME ACTIVATION

Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.

Synergistic enzyme mechanisms and effects of sequential enzyme additions on degradation of water insoluble wheat arabinoxylan

Generation of a fermentable hydrolysate from arabinoxylan is a first prerequisite in the utilization of wheat hemicellulose in the biofuel industry. This study examined the efficacy of four hemicellulolytic microbial enzyme preparations and one xylanase preparation in catalyzing degradation of purified water soluble and water insoluble wheat arabinoxylan—with particular emphasis on the catalytic degradation of water insoluble arabinoxylan. The effects of individual enzyme treatments were compared by assessing yields of arabinose, xylose, and xylobiose obtained under different reaction conditions of pH and temperature in response surface designs. In general, the monosaccharide yields obtained were lower on the water insoluble wheat arabinoxylan than on the water soluble. On both substrates, the Ultraflo L preparation from Humicola insolens was best in catalyzing arabinose and xylobiose release, while the Celluclast 1.5 L preparation from Trichoderma reesei was superior to all the other enzyme preparations in catalyzing xylose release. Treatments with 50:50 combinations of the enzyme preparations only resulted in a pronounced synergistic xylose release with a mixture of Ultraflo L:Celluclast 1.5 L. Examination of the progress of the substrate degradation indicated that the synergism on the insoluble arabinoxylan resembled what we have previously observed on soluble arabinoxylan, i.e. that the effect was a result of positive interaction in arabinose release and xylan depolymerization between the α- l-arabinofuranosidase (EC 3.2.1.55) and endo-1,4-β-xylanase (EC 3.2.1.8) activities present in Ultraflo L, and between the β-xylosidase (EC 3.2.1.37) activity present in the Celluclast 1.5 L. The results of treatments with combinations of Ultraflo L and purified T. reesei β-xylosidase – with both simultaneous and sequential addition, and with and without contemporary pH adjustments to optimize individual enzyme activities – strongly corroborated this conclusion.

RETRACTED: Validation and application of a high-performance liquid chromatographic-based assay for determination of the inosine 5′-monophosphate dehydrogenase activity in erythrocytes

Thiopurine drug monitoring has become an important issue in treating children with acute lymphoblastic leukaemia (ALL). In this population, a genetic polymorphism causes wide differences in the activity of thiopurine S-methyletransferase (TPMT)--the rate-limiting enzyme of the thiopurine degradation metabolism--leading to the necessity of drug dose adjustments. It is not yet known if similar differences exist in the inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of the thiopurine synthesis. To test this, we established and validated a high-performance liquid chromatographic (HPLC)-based assay to determine the IMPDH enzyme activity in erythrocytes. The remarkable features of this assay are its simple erythrocyte separation/haemolysis and assay conditions and a distinct segregation of xanthosine 5'-monophosphate (XMP) from the clear supernatant after precipitation. The probes were processed without a time-consuming extraction and heating procedure and the assay demonstrated a good intra- and interday stability as well as a recovery rate of approximately 100%. The IMPDH enzyme activity was measured in erythrocytes of 75 children with diagnosis of ALL before starting antileukaemic therapy and their activity compared to those of 35 healthy adult controls. The measured enzyme activity was wide ranging in both groups. The individual enzyme activity differences observed in children with ALL might led to differences in the thionucleotide levels in those undergoing the standard thiopurine dose regimen.

Scaling of Na+,K+‐ATPase Molecular Activity and Membrane Fatty Acid Composition in Mammalian and Avian Hearts

We have examined Na(+),K(+)-ATPase molecular activity and membrane fatty acid composition in the heart of six mammalian and eight avian species ranging in size from 30 g in mice to 280 kg in cattle and 13 g in zebra finches to 35 kg in emus, respectively. Na(+),K(+)-ATPase activity scaled negatively with body mass in both mammals and birds. In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and molecular activity (turnover rate) of Na(+),K(+)-ATPase enzymes, while in small birds, higher Na(+),K(+)-ATPase activity appeared to result primarily from an increased molecular activity of individual enzymes. The unsaturation index of cardiac phospholipids scaled negatively with body mass in both groups, while a significant allometric increase in monounsaturate content was observed in the larger mammals and birds. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid (22:6n-3) displayed the greatest variation, scaling negatively with body mass and varying greater than 40-fold in both mammals and birds. Membrane fatty acid profile was correlated with Na(+),K(+)-ATPase molecular activity in both mammals and birds, suggesting a potential association between membrane lipid composition and the activity of membrane-bound enzymes in the hearts of endotherms.

Extent of ipt gene expression and resulting amount of cytokinins affect activities of carboxylation enzymes in transgenic plants

Three types of transgenic plants of Solanum tuberosum cvs. Kamyk and Oreb, and Nicotiana tabacum cvs. Maryland Mammoth and Trapezond were selected according to intensity of introduced ipt gene expression and resulting amount of synthesised cytokinins (CKs). In comparison with controls, original transgenic regenerants grown in vitro showed a massive increase of CK contents, in tobacco by 379 % and in potato by 159 % (MAS). Potato grown in soil from tubers of transgenic plants demonstrated a moderate increase (44 %) of CK contents (MOD). Transgenic tobacco grown from seeds in vitro did not show any significant change in CK contents (NOT). Initial (RuBPCi and RuBPOi) and total (RuBPCt) activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and the activity of phosphoenolpyruvate carboxylase (PEPC) were not significantly affected by the transformation in the NOT plants. In the MOD plants, the RuBPCO activities were stimulated by up to 34 % whereas the PEPC activity was decreased by 17 %. On the other hand, all the measured enzyme activities were 32 - 91 % lower in the MAS. Leaf area, fresh and dry masses, and chlorophyll and soluble protein contents also went down with increasing CK amounts in the transformants. Dependence of RuBPCi/RuBPOi and RuBPCt/PEPC ratios on the relative CK amounts in transgenic plants revealed that the individual enzyme activities were not affected uniformly. Endogenous CK contents in the MAS thus apparently exceeded an optimum needed for positive effects on many physiological traits and became a stress factor for such plants.

Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodoptera frugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.

The Physiological Role of the Ribulose Monophosphate Pathway in Bacteria and Archaea

3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of the ribulose monophosphate pathway. This pathway, which was originally found in methylotrophic bacteria, is now recognized as a widespread prokaryotic pathway involved in formaldehyde fixation and detoxification. Recent progress, involving biochemical and genetic approaches in elucidating the physiological functions of HPS and PHI in methylotrophic as well as non-methylotrophic bacteria are described in this review. HPS and PHI orthologs are also found in a variety of archaeal strains. Some archaeal HPS orthologs are fused with other genes to form single ORF (e.g., the hps-phi gene of Pyrococcus spp. and the faeB-hpsB gene of Methanosarcina spp). These fused gene products exhibit functions corresponding to the individual enzyme activities, and are more efficient than equivalent systems made up of discrete enzymes. Recently, a novel metabolic function for HPS and PHI has been proposed in which these enzymes catalyze the reverse reaction for the biosynthesis of pentose phosphate in some archaeal strains. Thus the enzyme system plays a different role in bacteria and archaea by catalyzing the forward and reverse reactions respectively.

Diversity in the Activity of Individual Enzymes

Although the structure of an enzyme is often depicted as static, it is dynamic. Hence, a population of chemically identical enzymes has not one, but a distribution of structures at any moment in time. Does this have an effect on the activity of the enzyme? This article reviews experiments designed to test the hypothesis that this distribution of structures results in a distribution of enzyme activities. The experiments reviewed here use different enzymes, falvin adenine dinucleotide, beta-galactosidase, alkaline phosphatase, exonuclease I, lactate dehydrogenase I, alpha-chymotrypsin, the 20S proteasome, and horseradish peroxidase. All experiments come to the same conclusion, when measured individually, apparently identical enzymes show a distribution in rates of activity.

Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts

Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial. We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities. A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%-18%. With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.

Regulation and Seasonal Dynamics of Extracellular Enzyme Activities in the Sediments of a Large Lowland River

We tested whether seasonal changes in the sources of organic substances for microbial metabolism were reflected changes in the activities of five extracellular enzymes in the eighth order lowland River Elbe, Germany. Leucine aminopeptidase showed the highest activities in the water column and the sediments, followed by phosphatase > beta-glucosidase > alpha-glucosidase > exo-1,4-beta-glucanase. Individual enzymes exhibited characteristic seasonal dynamics, as indicated by their relative contribution to cumulative enzyme activity. Leucine aminopeptidase was significantly more active in spring and summer. In contrast, the carbohydrate-degrading enzymes peaked in autumn, and beta-glucosidase activity peaked once again in winter. Thus, in sediments, the ratio of leucine aminopeptidase/beta-glucosidase reached significant higher medians in spring and summer (5-cm depth: ratio 7.7; 20-cm depth: ratio 10.1) than in autumn and winter (5-cm depth: ratio 3.7, 20-cm depth: ratio 6.3). The relative activity of phosphatase in the sediments was seasonally related to both the biomass of planktonic algae as well as to the high content of total particulate phosphorus in autumn and winter. Due to temporal shifts in organic matter supply and changes in the storage capacity of sediments, the seasonal peaks of enzyme activities in sediments exhibited a time lag of 2-3 months compared to that in the water column, along with a significant extension of peak width. Hence, our data show that the seasonal pattern of extracellular enzyme activities provides a sensitive approach to infer seasonal or temporary availability of organic matter in rivers from autochthonous and allochthonous sources. From the dynamics of individual enzyme activities, a consistent synoptic pattern of heterotrophic functioning in the studied river ecosystem could be derived. Our data support the revised riverine productivity model predicting that the metabolism of organic matter in high-order rivers is mainly fuelled by autochthonous production occurring in these reaches and riparian inputs.

Perinatal lethal phenotype with generalized ichthyosis in a type 2 Gaucher disease patient with the [L444P;E326K]/P182L genotype: Effect of the E326K change in neonatal and classic forms of the disease

Gaucher disease, the most common lysosomal storage disorder, encompasses a wide spectrum of clinical symptoms. The perinatal lethal form is very rare and is considered a distinct form of classic type 2 Gaucher disease. Prominent features of the severe perinatal form are hepatosplenomegaly variable, associated with hydrops fetalis and ichthyosis. Here, we describe a child who presented generalized ichthyosis and died at 25 days of age. Genotype analysis revealed compound heterozygosity for the complex allele [L444P;E326K] and mutation P182L, described for the first time in this patient. Mutations E326K and L444P were on the same chromosome. Expression studies of mutant glucocerebrosidases showed that the double mutant allele had lower activity, 8.5% of wild type, in contrast to the activity of individual E326K and L444P mutant enzymes, 42.7% and 14.1%, respectively. The P182L mutant enzyme showed no glucocerebrosidase activity. A revision of the genotypes identified in a series of Spanish patients with type 2 Gaucher disease showed that the complex allele [L444P;E326K] accounted for 19.2% of patient alleles and that homozygosity for this allele or its heterozygosity with mutation L444P, or another severe mutation such as P182L, was associated with the perinatal lethal presentation of the disease. In contrast, the [L444P;E326K] allele was not detected in patients with classic type 2 diagnosed when several months old. The high frequency of the E326K substitution observed in patients with type 2 as compared to the general population (0.5%) suggests that this change may have a modulating negative effect on the clinical condition of these Gaucher disease patients when present in combination with mutation L444P. The relatively high prevalence of the double mutant allele in Spanish patients prompted us to perform a haplotype analysis, using four polymorphic markers, which suggest a common origin for this allele. During the mutational analysis of the series of type 2 patients, a novel mutation, I260T (c.896T > C), was identified.