Glycogen Debranching Enzyme Research Articles

植物のデンプン合成代謝システムに関する一考察：イネはい乳を中心に

The tandem-cluster structure of amylopectin must be a refined product that higher plants have evolved from the cluster-free random branched structure of glycogen. During the evolutionary process, plants have increased the number of isozymes of three classes of enzymes, namely soluble starch synthase (SS), starch branching enzyme (BE) and starch debranching enzymes (DBE), by giving each individual enzyme a distinct function in constructing the amylopectin cluster structure. On the basis of biochemical and molecular analyses of starch mutants and transgenic plants of rice, the present paper presents a model for amylopectin cluster biosynthesis in rice endosperm to understand how each enzyme is involved in this process. The author also proposes a notion called “cluster-world” where by the structure of amylopectin varies through changes in several distinct patterns of the cluster structure, and alteration of activities of key enzymes such as SSIIa, BEIIb and isoamylase1 results in the structural variation within the fixed pattern depending on which enzyme activity is altered.

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan.

To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.

Characterization of a SINE indel polymorphism in the porcine AGL gene and assignment of the gene to chromosome 4q.

*Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic. Institute of Animal Husbandry and Breeding, Department of Animal Breeding and Biotechnology, University of Hohenheim, Garbenstrasse 17, D-70593 Stuttgart, Germany. Ghent University, Faculty of Veterinary Medicine, Department of Animal Nutrition, Genetics, Breeding and Ethology, Heidestraat 19, 9820 Merelbeke, Belgium. DIPROVAL Sezione di Allevamenti Zootecnici, Faculty of Agriculture, University of Bologna, Via F.lli Rosselli 107, 42100 Reggio Emilia, Italy

One- and Two-dimensional Native PAGE Activity Gel Analyses of Maize Endosperm Proteins Reveal Functional Interactions between Specific Starch Metabolizing Enzymes

Starch metabolizing enzyme isoforms are known to exhibit functional interactions. This research employed one and twodimensional starch zymograms to investigate pleiotropic enzymatic effects in starch branching enzyme (BE) and debranching enzyme (DBE) mutants of maize. Activity bands are shown to correspond with specific gene products by immunoblot analysis or biochemical assay. Multiple migratory forms are observed for BEI and BEIIa, and a single form for BEIIb. SU1 isoamylase-type DBE migrates as three distinct forms and ZPU1 pullulanase-type DBE as a single form. For each BE and DBE, a genetic null mutation conditions complete loss of all forms of the respective enzyme. The BE mutation amylose-extender also results in loss of two BEI migration forms, and the sul-starchy and zpul-204 DBE mutations result in a loss of BEIIa activity. In both DBE mutants, BEIIa is of normal abundance and size, indicating the effect is posttranslational and directly impacts enzyme function. Zymogram analysis of the uncharacterized opaque5 (o5) mutant shows o5 endosperm is deficient in β-amylase activity, providing insight into a biochemical defect resulting from the mutation. Finally, a novel enzyme activity identified in starch zymograms was characterized and found to be a glucan hydrolase of the β-amylase type. This research demonstrates that activity gel analysis is an effective approach for uncovering pleiotropic enzymatic effects that might not be discovered using RNA microarray or proteomics technologies, and indicates that some mutational effects on starch metabolizing enzymes are likely due to altered protein complex associations.

Some Biochemical Aspects of the Biosynthesis of Glycogen and Amylopectin

Abstract: Whilst it is generally accepted that glycogen is synthesized from UDPG by the combined action of a chain-lengthening enzyme (glycogen synthase) and a branching enzyme (BE), the synthesis of amylopectinfrom ADPG requires a more complex enzyme system which includes various isoforms of the related starch synthase and plant BEs. In addition, several workers have suggested that debranching enzymes (DBEs) are involved. These suggestions, which are based mainly on molecular biological experiments, require mechanisms which are incompatible with much of the biochemical evidence. Overall, the involvement of DBEs in amylopectin biosynthesis has not been unambiguously established, although DBEs could play a role in the control of phytoglycogen biosynthesis by a separate pathway, or in starch granule initiation and growth.

Genetic Analysis Indicates Maize Pullulanase- and Isoamylase-type Starch Debranching Enzymes Have Partially Overlapping Functions in Starch Metabolism

Genetic Analysis Indicates Maize Pullulanase- and Isoamylase-type Starch Debranching Enzymes Have Partially Overlapping Functions in Starch Metabolism

Structures and Properties of Amylopectin and Phytoglycogen in the Endosperm of sugary-1 Mutants of Rice

Starch debranching enzyme (DBE)-deficient, sugary-1 mutations of rice caused the biosynthesis of more highly branched α-glucan in the form of phytoglycogen and sugary -amylopectin rather than wild-type amylopectin. Phytoglycogen and sugary -amylopectin are located in the inner and outer regions of the rice endosperm, respectively. Detailed analyses of these glucans from a variety of allelic mutants with different severity of sugary phenotype revealed that sugary -amylopectins consisted of more short chains of DP<12 but fewer chains of DP13–24 than did wild-type amylopectin, and the alterations reflected on lower molecular weights, smaller gyration radii, and denser molecules. These changes resulted in lower onset gelatinization temperature, lower peak viscosity, lesser degree of crystallinity, and smaller and cracked granules with rough surface of sugary starch granules as compared with that of the wild-type. These results indicate that changes in the fine structure of amylopectin induced by lesion of DBE greatly influence the morphological and physicochemical properties of starch. Phytoglycogens from sugary-1 mutants were composed of multiple components of smaller molecular weights and gyration radii than sugary -amylopectin and amylopectin. The highly branched phytoglycogens were more tightly packed molecules exhibiting greater dispersed densities than sugary -amylopectin and amylopectin. These results suggest that the levels of DBE in rice endosperm control the biosynthetic pathway of branched α-glucans and produce amylopectin, sugary -amylopectin and phytoglycogen.

Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme

Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes α(1→6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, β-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.

Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue.

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.

Clinical and genetic variability of glycogen storage disease type IIIa: seven novel AGL gene mutations in the Mediterranean area.

Deficiency of amylo-1,6-glucosidase, 4-alpha-glucanotransferase enzyme (AGL or glycogen debrancher enzyme) is responsible for glycogen storage disease type III, a rare autosomal recessive disorder of glycogen metabolism. The AGL gene is located on chromosome 1p21, and contains 35 exons translated in a monomeric protein product. The disease has recognized clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different subjects. The clinical manifestations of GSD III are represented by hepatomegaly, hypoglycemia, hyperlipidemia, short stature and, in a number of subjects, cardiomyopathy and myopathy. In this article, we discuss the genotypic-phenotypic heterogeneity of GSD III by the molecular characterization of mutations responsible for the disease on a collection of 18 independent alleles from the Mediterranean area. We identified by heteroduplex band shift, DNA direct sequencing, and restriction analysis, seven novel mutations (four nonsense point-mutations: R34X, S530X, R1218X, W1398X; two microinsertions: 1072insT and 4724insAA; and one bp deletion: 676DeltaG), together with two new cases carrying a IVS21 + 1 G --> A splicing site mutation previously described in Italian patients. Altogether, 15 alleles were characterized. The correlation between type of mutation and clinical severity was studied in six patients in whom both mutated alleles were detected. Our data confirm the extreme genetic heterogeneity of this disease, thus precluding a strategy of mutation finding based on screening of recurrent common mutations.

Analysis of Mesorhizobium loti glycogen operon: effect of phosphoglucomutase (pgm) and glycogen synthase (g/gA) null mutants on nodulation of Lotus tenuis.

The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), beta cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophosphorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.

Identification of the Catalytic Residues of Bifunctional Glycogen Debranching Enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.

Combination of miglitol, an anti-diabetic drug, and nicorandil markedly reduces myocardial infarct size through opening the mitochondrial K(ATP) channels in rabbits.

The anti-diabetic drug miglitol, an alpha-glucosidase inhibitor, which is currently used clinically, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the alpha-1,6-glucosidase of glycogen-debranching enzyme in the heart. Nicorandil, a K(ATP) channel opener with a nitrate-like effect, which is also currently used clinically, also reduces the infarct size. Therefore, we hypothesized that combination of nicorandil and submaximal dose of miglitol could markedly reduce myocardial infarct size more than miglitol or nicorandil alone, and investigated the mechanism for the infarct size-reducing effect. Japanese white rabbits without collateral circulation were subjected to 30 min coronary occlusion followed by 48 h reperfusion. Pre-ischaemic treatment with submaximal dose of miglitol (5 mg kg(-1), i.v.) and nicorandil alone (100 microg kg(-1) min(-1) 5 min) moderately reduced the infarct size as a percentage of area at risk (24+/-4 and 25+/-4%, respectively), and 10 mg kg(-1) of miglitol markedly reduced the infarct size (15+/-2%) compared with the controls (42+/-2%). Combination of 5 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min), and 10 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min) significantly reduced the infarct size (13+/-4 and 12+/-3%, respectively) more than miglitol or nicorandil alone. Pretreatment with 5HD completely abolished the infarct size-reducing effect of 10 mg kg(-1) of miglitol alone (36+/-7%) and that of combination of 5 mg kg(-1) of miglitol and nicorandil (46+/-2%). Combination of nicorandil and submaximal dose of miglitol markedly reduced the myocardial infarct size more than miglitol or nicorandil alone. This effect was suggested to be related to the opening of mitochondrial K(ATP) channels.

Molecular genetic basis and prevalence of glycogen storage disease type IIIA in the Faroe Islands.

Glycogen storage disease type IIIA (GSD IIIA) is caused by mutations of the amyloglucosidase gene (AGL). For most populations, none of the AGL mutations described to date is particularly frequent. In this paper, we report that six children with GSD IIIA from the Faroe Islands were found to be homozygous for the novel nonsense mutation c.1222C>T (R408X) of the AGL gene. This mutation is easily detected by restriction enzyme digest with NsiI after mismatch PCR. Investigating five intragenic polymorphisms, we could show that this mutation was always associated with the same haplotype. The c.1222C>T mutation could be detected on two chromosomes of another 50 unselected GSD IIIA patients of other European or North American origin which means that this mutation plays a minor role worldwide. From the fact that we are currently aware of a total of 14 GSD IIIA cases in the Faroese population of 45 000, the observed prevalence is 1 : 3100. While the novel AGL mutation c.1222C>T was not detectable among 198 German newborns, nine out of 272 children from the Faroese neonatal screening program were found to be heterozygous for this mutation. Thus, the calculated prevalence is 1 : 3600 (95% CI 1:700-1:6400). We conclude that due to a founder effect, the Faroe Islands have the highest prevalence of GSD IIIA world-wide. The detection of the molecular defect has facilitated the diagnosis and has offered the opportunity for prenatal diagnosis in this patient group.

Combination of N-methyl-1-deoxynojirimycin and ischemic preconditioning markedly reduces the size of myocardial infarcts in rabbits.

N-methyl-1-deoxynojirimycin (NMDN), an a-glucosidase inhibitor, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the alpha-1,6-glucosidase of glycogen-debranching enzyme in the heart, in addition to possessing an antihyperglycemic action by blocking alpha-1,4-glucosidase in the intestine. Ischemic preconditioning (PC), which markedly reduces the size of the myocardial infarct, is known to reduce the activity of phosphorylase and reduce the glycogenolytic rate. Therefore, it was hypothesized that a combination of pharmacological inhibition of glycogenolysis by an alpha-1,6-glucosidase inhibitor, NMDN, and PC could markedly reduce myocardial infarct size more than NMDN or PC alone. Japanese white rabbits without collateral circulation were subjected to a 30-min coronary occlusion followed by 48-h reperfusion. The infarct sizes as a percentage of area at risk were significantly reduced by pre-ischemic treatment with either 100mg/kg of NMDN or PC of 5 min ischemia and 5 min reperfusion alone (15.9+/-2.0%, n=8, and 10.3+/-1.2%, n=8, respectively) as compared with the control (43.9+/-2.2%, n=8). However, the combination of 100mg/kg of NMDN and PC significantly reduced the infarct size (4.9+/-1.2, n=8) compared with NMDN or PC alone. Another 40 rabbits, also given 100mg of NMDN, PC, NMDN+PC or saline before ischemia (n=10 in each group), were killed for biochemical analysis after 30 min of ischemia. NMDN and PC preserved the glycogen content and attenuated the lactate accumulation, respectively, as compared with the control. However, the combination of NMDN and PC preserved significantly more glycogen and significantly reduced lactate accumulation than either NMDN or PC alone. The combination of NMDN and PC markedly reduced the myocardial infarct size more than either process alone. The marked preservation of glycogen and marked attenuation of lactate accumulation by the combination of NMDN and PC suggest that the mechanism for this effect of NMDN+PC is related to the inhibition of glycogenolysis.

The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

The Saccharomyces cerevisiaeYPR184w gene encodes the glycogen debranching enzyme

The YPR184w gene encodes a 1536-amino acid protein that is 34–39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the α-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of α-1,4-glucanotransferase and α-1,6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The α-1,4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, markedly improves postischemic left ventricular dysfunction.

We examined whether pharmacological inhibition of glycogenolysis by N-methyl-1-deoxynojirimycin (MOR-14), a new compound which reduces the glycogenolytic rate by inhibiting the alpha-1,6-glucosidase activity of the glycogen-debranching enzyme, can protect the heart against postischemic left ventricular dysfunction. The hearts of male Sprague-Dawley rats were excised, and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. The hearts were paced at 320 beats/min except during the ischemia. Left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s), and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min including a 30-min preischemic period followed by a 30-min episode of global ischemia and 60 min reperfusion. with or without 0.5 or 2 mM of MOR-14 during the 30-min preischemic period or the first 30 min of reperfusion. In another series of experiments, the myocardial content of glycogen and lactate was measured during the 30-min episode of ischemia in groups treated with and without 2mM of MOR-14. Preischemic but not postischemic treatment with MOR-14 significantly improved LVDP and +/-dP/dt without altering coronary flow during reperfusion in a dose-dependent manner. MOR-14 significantly preserved the glycogen content and significantly attenuated the lactate accumulation during the 30-min episode of ischemia. Preischemic treatment with MOR-14 is protective against postischemic left ventricular dysfunction through the inhibition of glycogenolysis in the isolated rat heart.

High Expression of Glycogen-Debranching Enzyme in Escherichia coli and Its Competent Purification Method

Glycogen-debranching enzyme (GDE) gene from Saccharomyces cerevisiae was cloned and expressed into Escherichia coli. A 99.3% homology was found between the nucleotide sequences of GDE gene harbored in the recombinant E. coli plasmid (pTrc99A) and the open reading frame (902039–906646 position) of the 4608-bp fragment of S. cerevisiae chromosome XVI. We investigated the best conditions for GDE expression. When the cultivation temperature of recombinant E. coli strains was lowered to 25°C and the isopropyl-β-d-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from a liter culture as estimated by amylo-1,6-glucosidase activity. Consecutively, we developed a new method for purifying GDE. The method requires only a single-step purification via β-cyclodextrin-immobilized Sepharose 6B (β-CD Sepharose 6B) affinity chromatography and renders a 90% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. With the highly expressed GDE in recombinant E. coli and a rapid and effective purification method, we successfully resolved the hurdle always faced for obtaining an ample amount of purified GDE. The availability of GDE, hence, may allow advancement on GDE studies and provide new prospects for GDE on biotechnological application.

Effects of Aerobic and Anaerobic Germination on the Debranching Enzyme, Limit Dextrinase, in Barley Malt

Limit dextrinase exists in an active free form and in a soluble inhibited (latent) form. The total activity of limit dextrinase is measured after extracting the enzyme in the presence of dithiothreitol, and the free enzyme is measured following extraction without dithiothreitol. Limit dextrinase is normally in the inhibited form following malting. The activity of limit dextrinase in grains of barley, Hordeum vulgare L, variety Golden Promise, germinated using combinations of aerobic and anaerobic conditions, was studied. The total limit dextrinase measurable with dithiothreitol in the extraction increased with increasing days of aerobic germination until it came close to a plateau after six days. The transformation of this inhibited enzyme to the free and uninhibited enzyme required three to four days of anaerobic germination. The effect of an extract from an aerobically germinated malt on the uninhibited enzyme of an anaerobically germinated malt was studied. Mixing increasing amounts of aerobic malt (free limit dextrinase activity of 60 mU/g) with anaerobic malt led to the inhibition of the free enzyme in the anaerobic malt. The inhibition increased rapidly as the proportion of aerobic to anaerobic malt approached a ratio of 1:1. Thereafter, increasing amounts of aerobic malt only slightly decreased the small amount of residual free limit dextrinase activity remaining. Thus, in a mash, excess inhibitor from a normal malt would almost certainly inhibit free enzyme from an anaerobic malt.