Acid Alpha-glucosidase Activity Research Articles

Effect of ammonia on endocytosis, cytokine production and lysosomal enzyme activity of a microglial cell line

Ammonia is a natural lysosomotropic compound. Concentrations of ammonium acetate > 2 mM impaired the phagocytic activity of BV-2 cells, an immortalized microglial cell line, as was determined by the uptake of fluorescent latex microspheres of different sizes. In contrast, an increase in the uptake of fluorescent dextran was observed with the elevation in ammonium acetate concentrations. This indicates that ammonia affects phagocytotic and pinocytotic activities of BV-2 cells differently. Interferon-gamma- and polyinosinic-polycytidylic acid-stimulated secretion of IL1 alpha as well as LPS-stimulated secretion of IL6 decreased with an elevation in ammonium acetate concentrations. The constitutive secretion of IL1 alpha was not significantly affected by ammonium acetate. However, an increase in LPS-stimulated IL1 alpha secretion was observed at 10 mM and 20 mM ammonium acetate. High concentrations of ammonia affected the activity of lysosomal enzymes of the BV-2 cells. Acid phosphatase and alpha-glucosidase activities increased with the increase in ammonium acetate up to 20 mM. The activity of cathepsin D was increased at 5 mM, but decreased at higher ammonia concentrations. The effects of ammonia on microglial functions are discussed with respect to pathogenetic mechanisms of dementia of the Alzheimer type.

Islet glucan-1,4-alpha-glucosidase: differential influence on insulin secretion induced by glucose and isobutylmethylxanthine in mice.

In previous in-vivo studies we have presented indirect evidence for the involvement of islet acid glucan-1,4-alpha-glucosidase (acid amyloglucosidase), a lysosomal glycogen-hydrolysing enzyme, in certain insulin secretory processes. In the present combined in-vitro and in-vivo investigation, we studied whether differential changes in islet acid amyloglucosidase activity were related to the insulin secretory response induced by two mechanistically different secretagogues, glucose and isobutylmethylxanthine (IBMX). It was observed that addition of the selective alpha-glucosidehydrolase inhibitor emiglitate (1 mmol/l) to isolated pancreatic islets resulted in a marked reduction of glucose-induced insulin release. This was accompanied by a pronounced suppression of islet activities of acid amyloglucosidase and acid alpha-glucosidase, whereas other lysosomal enzyme activities, such as acid phosphatase and N-acetyl-beta-D-glucosaminidase, were unaffected. Furthermore, islets first incubated with emiglitate in the presence of high (16.7 mmol/l) glucose released less insulin than untreated controls in response to glucose in a second incubation period in the absence of emiglitate. In contrast, IBMX-induced insulin release was not influenced by emiglitate although accompanied by a marked reduction of islet activities of all three alpha-glucosidehydrolases. Basal insulin secretion (1 mmol glucose/l) was unaffected in the presence of emiglitate. In-vivo pretreatment of mice with highly purified fungal amyloglucosidase ('enzyme replacement'), a procedure known to increase islet amyloglucosidase activity, resulted in a greatly enhanced insulin secretory response to an i.v. glucose load. The increase in insulin release was accompanied by a markedly improved glucose tolerance curve in these animals. In contrast, enzyme pretreatment did not influence the insulin response or the blood glucose levels after an i.v. injection of IBMX. The data lend further support to our hypothesis that islet acid amyloglucosidase is involved in the multifactorial insulin secretory processes induced by glucose but not in those involving direct activation of the cyclic AMP system. The results also indicate separate, or at least partially separate, pathways for insulin release induced by glucose and IBMX.

Islet lysosomal enzyme activities and glucose-induced insulin secretion: effects of mannoheptulose, 2-deoxyglucose and clonidine.

In previous investigations we have shown a striking relationship between the activity of glycogenolytic glucose producing acid hydrolases in pancreatic islet tissue and certain insulin-releasing processes. In the present investigation we have studied the relation between islet lysosomal enzyme activities and glucose-induced insulin secretion in vitro in the presence of various insulin secretory inhibitors. It was observed that the nonmetabolizable glucose analogue, mannoheptulose (5 mmol/l) did induce a 2-fold increase in insulin release at low (1 mmol/l) glucose, and a total suppression of insulin release at high (16.7 mmol/l) glucose. These changes in the insulin-secretory pattern were accompanied by similar changes in the activity of islet acid alpha-glucosidase. The activities of neutral alpha-glucosidase (endoplasmic reticulum) or acid phosphatase and N-acetyl-beta-D-glucosaminidase (lysosomes) were not affected by mannoheptulose. 2-Deoxyglucose (5 mmol/l), another glucose analogue, did not increase insulin secretion or acid alpha-glucosidase activity at low glucose. At high glucose, however, a partial inhibition of both insulin release (approximately 50%) and acid alpha-glucosidase activity was seen. 2-Deoxyglucose slightly suppressed acid phosphatase activity but did not influence the activities of neutral alpha-glucosidase or N-acetyl-beta-D- glucosaminidase. Direct addition of glucose to islet homogenates showed a suppressive effect on alpha-glucosidase activity at pH 4.0 and 5.0. The glucose analogues displayed only marginal (-10%) inhibition of alpha-glucosidase activity at pH 5.0. No effect of the analogues was seen at pH 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of islet amyloglucosidase activity and glucose-induced insulin secretion.

We have previously presented evidence for the involvement of islet acid amyloglucosidase, a lysosomal glycogen-hydrolyzing enzyme, in certain insulin secretory processes. In the present investigation, we studied whether differential changes in islet amyloglucosidase activity could be related to the insulin secretory response to glucose. It was observed that the dose-response curve for glucose-induced insulin response in vivo was shifted to the left by pretreatment of mice with purified fungal amyloglucosidase. In enzyme-pretreated mice, the ED50 was 2.1 mmol/kg glucose as compared with 5.7 mmol/kg in saline-pretreated controls (p less than 0.005). Also, the maximal insulin response to glucose was enhanced by amyloglucosidase pretreatment. Parenteral administration to mice (four injections during 2 days) of the pseudotetrasaccharide acarbose, a recognized inhibitor of intestinal alpha-glucosidases, surprisingly induced a marked increase in the activities of islet acid amyloglucosidase (+ 120%; p less than 0.001) and acid alpha-glucosidase (+ 45%; p less than 0.01) without affecting the activities of other lysosomal enzymes such as acid phosphatase and N-acetyl-beta-D-glucosaminidase. No effect on the microsomal neutral alpha-glucosidase was recorded. Moreover, in these mice, the insulin secretory response to glucose was enhanced both at a maximal dose of glucose 11.1 mmol/kg and at a dose in the ED25-ED50 range, 3.3 mmol/kg (p less than 0.005). Direct addition of acarbose to islet homogenates strongly suppressed acid amyloglucosidase activity, the EC50 being approximately 1 microM. Acid alpha-glucosidase activity was also strongly inhibited, whereas the activities of acid phosphatase and N-acetyl-beta-D-glucosaminidase were unaffected. Neutral alpha-glucosidase was slightly suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Human acid maltase-deficient myogenic cell transformation with origin-defective SV40: Characterization of a cloned line

A clonal human skeletal muscle cell line showing acid maltase deficiency (AMD) was established through the transfection of origin-defective SV40 DNA. The low acid alpha-glucosidase activity and glycogenosomes in this clone corresponded to AMD. This clone, in spite of loading glycogenososmes, was competent not only as to proliferation without contact inhibition but also as to myogenic differentiation to some extent. Dexamethasone promoted the formation by the transformant of multinucleated myotubes, which expressed acetylcholine receptors. The existence of glycogenosomes did not seem to affect the proliferation or differentiation of myoblasts. The aberrant acid alpha-glucosidase expressed in the transformed myogenic clone was shown to be biochemically identical to that in AMD fibroblasts. This transformant should be of great value for investigating the pathogenesis of AMD because of the possibility of supplying semi-permanently a uniform myogenic cell line expressing AMD.

Intravenous administration of phosphorylated acid alpha-glucosidase leads to uptake of enzyme in heart and skeletal muscle of mice.

The lysosomal storage disorder glycogenosis type II is caused by acid alpha-glucosidase deficiency. In this study we have investigated the possible applicability of mannose 6-phosphate receptor-mediated enzyme replacement therapy to correct the enzyme deficiency in the most affected tissues. Bovine testes acid alpha-glucosidase containing phosphorylated mannose residues was intravenously administered to mice and found to be taken up by heart (70% increase of activity) and skeletal muscle (43% increase); the major target organs. The uptake of nonphosphorylated human placenta acid alpha-glucosidase by heart and skeletal muscle appeared to be significantly less efficient, whereas uptake of dephosphorylated bovine testes enzyme was not detectable. The phosphorylated bovine testes acid alpha-glucosidase remained present in mouse skeletal muscle up to 9-15 d after administration, with a half-life of 2-4 d. Besides being measured in skeletal muscle and heart, uptake of phosphorylated bovine testes and nonphosphorylated human placenta acid alpha-glucosidase was measured in several other organs, but not in brain. The increase of acid alpha-glucosidase activity was highest in liver and spleen. We concluded that application of mannose 6-phosphate receptor-mediated enzyme replacement therapy may offer new perspectives for treatment of glycogenesis type II.

Natural bone marrow transplantation in cattle with Pompe's disease

Adding acid alpha-glucosidase to cultures of Pompe's disease muscle has resulted in enzyme uptake and reduction in concentration of glycogen. However, bone marrow transplantation has been unsuccessful as a treatment. Immune rejection may have contributed to this failure. Twin calves share a placenta and carry lymphoreticular cells of each other's type, they become lymphoreticular chimeras in utero and immune rejection does not occur. One natural and three sets of twins produced by embryo transfer were studied in Pompe's disease cattle. Chimerism persisted throughout life and the situation was analogous to a transplant of histocompatible bone marrow stem cells. The activity of acid alpha-glucosidase in leucocytes and in biopsies of the semitendinosus muscle and the mean activity in diaphragm, spleen and lymph node obtained after death from affected twins were significantly higher than in single affected calves. Glycogen concentration was lowered in liver, spleen and lymph node but not in muscles. The affected twins showed clinical signs and changes in muscle similar to those seen in affected single calves. It is concluded that bone marrow transplantation is unlikely to be a successful treatment for Pompe's disease.

Effect of the Lysosomotropic Drug Suramin on Islet Lysosomal Enzyme Activities and the Insulin-Secretory Response Induced by Various Secretagogues

The trypanocidal drug suramin is known to concentrate in lysosomes and to depress the activity of different lysosomal enzymes. We have previously shown that suramin can inhibit the activity of the islet lysosomal enzyme acid amyloglucosidase, a glycogenolytic glucose-producing hydrolase, which seems to be involved in certain insulin-secretory processes. In the present investigation we studied the pH dependency and dose-response effects of suramin on islet lysosomal enzyme activities as well as the effect of suramin treatment on the insulin-secretory response to various secretagogues in mice. It was found that two injections of suramin (0.18 mmol/kg) to normal NMRI mice at -24 and -2 h induced a moderate depression of the activities of islet acid amyloglucosidase (-22%) and acid phosphatase (-13%), whereas no effect was recorded for the activities of acid alpha-glucosidase, N-acetyl-beta-D-glucosaminidase and the non-lysosomal enzyme neutral alpha-glucosidase. Direct addition of different concentrations of suramin to islet homogenates showed that the drug was a potent inhibitor of acid amyloglucosidase and acid alpha-glucosidase at pH 4.0. At pH 5.0, suramin induced a large increase in acid alpha-glucosidase activity, whereas acid amyloglucosidase and acid phosphatase were inhibited. Suramin-injected mice showed a reduced insulin-secretory response to the sulphonylurea drug glibenclamide (-45%), whereas the insulin response to the cholinergic agonist carbachol or the phosphodiesterase inhibitor IBMX (1-isobutyl-3-methylxanthine) was unaffected. It is concluded that suramin inhibits islet acid amyloglucosidase activity in vivo and in vitro, whereas its effect on acid alpha-glucosidase is complex and pH dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of active human urokinase-type plasminogen activator in the milk of transgenic mice

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. Genomic DNA PCR and Northern blot analysis were used to determine the integration and mRNA expression of the hGAA gene in the putative transgenic rice cells. SDS-PAGE and Western blot analysis showed that the glycosylated precursor recombinant hGAA had a molecular mass of 110 kDa due to the presence of seven N-glycosylation sites. The accumulation of hGAA protein in the culture medium was approximately 37 mg/L after 11 days of culturing in a sugar depletion medium. The His tagged-hGAA protein was purified using an Ni-NTA column and confirmed as the precursor form of hGAA without the signal peptide encoded by the cDNA on the N-terminal amino acid sequence. The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells.

Heat-stable protein that stimulates acid alpha-glucosidase.

A hot-water extract of bovine spleen and guinea pig liver exhibited the ability to enhance acid alpha-glucosidase activity, with methylumbelliferyl alpha-glucoside, glycogen or maltose as substrate. The level of activator required for maximal stabilization was similar for all three substrates, indicating direct action on the enzyme rather than on substrate. The stimulator was partially purified by chromatography with gel-permeation (apparent Mr 20,000-24,000), ion-exchange and C4 reverse-phase columns. It was retained by a narrow-pore dialysis tubing and destroyed by treatment with Pronase, and is presumably a protein. The stimulating protein protected the enzyme against denaturation by heat or incubation with a buffer of high ionic strength in the absence of substrate. RNA inhibited the enzyme, and the activator protein was able to counteract the effect. Activating material was found in a variety of mouse and rat tissues, as well as human urine.

Insulin secretion and islet lysosomal enzyme activities in the mouse

To study the functional role of the lysosomes in islet physiology, we have in the present study investigated influences on insulin secretion induced by a primary inhibition of islet lysosomal enzyme activities by the use of the lysosomotropic drug suramin. First, we demonstrated that the activities of the three lysosomal enzymes, acid amyloglucosidase, acid alpha-glucosidase, and N-acetyl-beta-D-glucosaminidase, were inhibited in mouse islet homogenates upon direct addition of suramin (p less than 0.001). Thereafter, we studied the influences of suramin on islet lysosomal enzyme activities at 24 h after an administration of suramin to mice. We thereby found that the islet lysosomal enzyme activities were not significantly altered compared to controls. However, after incubation of the islets at 3.3 mM glucose, the activities of acid amyloglucosidase and acid alpha-glucosidase were increased (p less than 0.05). The activity of N-acetyl-beta-D-glucosaminidase was, however, not affected. Hence, the direct inhibitory action on islet lysosomal enzyme activities by suramin seems to be counteracted in vivo. Concurrently with the increased enzyme activities in incubated islets, glucose-induced insulin secretion was significantly potentiated by suramin pretreatment (p less than 0.01). In contrast, suramin pretreatment did impair the glucose-induced insulin secretion in vivo (p less than 0.01). The direct effects on plasma insulin and glucose levels of acutely administered suramin were also studied. It was found that at a high dose level, suramin slightly reduced the basal plasma insulin levels (p less than 0.05). Suramin did not, however, affect the glucose-induced increase in plasma insulin levels. Conversely, at a low dose level, suramin potentiated glucose-induced increase in plasma insulin levels (p less than 0.01). A similar potentiated glucose-induced insulin secretion by suramin was also observed by a direct addition of the drug to islets in vitro. In conclusion, this study demonstrates that suramin inhibits lysosomal enzyme activities in islet homogenates, increases the activity of acid amyloglucosidase and acid alpha-glucosidase in incubated mouse islets after administration of the drug in vivo and potentiates glucose-stimulated insulin secretion from islets in vitro after administration of the drug in vivo. Thus, we suggest first, that in vivo, compensatory mechanisms may counteract the direct inhibitory influence of suramin on islet lysosomal enzyme activities, and second, that in vitro, islet lysosomal acid amyloglucosidase and acid alpha-glucosidase activities and glucose-induced insulin secretion are regulated in parallel.

Prospect for enzyme therapy in glycogenosis II variants: a study on cultured muscle cells.

Impairment of skeletal muscle function is the common feature of distinct clinical forms of glycogenosis type II. In the present study, muscle cultures from different patients were used to investigate the cause of clinical heterogeneity and the feasibility of enzyme replacement therapy. The activity of acid alpha-glucosidase appears to be the primary factor in determining the extent of lysosomal glycogen storage in muscle, and thereby the clinical severity of the disease. Neutral alpha-glucosidases do not seem influential. Correction of the enzymatic defect was achieved in skeletal muscle cultures from patients by administration of a "high-uptake" form of acid alpha-glucosidase, purified from human urine. The enzyme reaches the lysosomes, including the glycogen storage vacuoles, and the lysosomal glycogen content is reduced to control level. In normal muscle cells 20% of the total cellular glycogen pool is segregated in lysosomal compartments. This percentage is higher than in fibroblasts, which may partly explain why muscles are more prone to store glycogen. The relevance of this study for enzyme therapy is discussed.

Km mutant of acid α‐glucosidase in a case of cardiomyopathy without signs of skeletal muscle involvement

A male patient is reported with a mutation of acid alpha-glucosidase causing an altered Km toward natural substrates. Cardiac arrhythmia was found at 12 years of age, and he died of heart failure at 15 years. No skeletal muscle involvement was observed either clinically or histologically. Acid alpha-glucosidase activity in fibroblasts was moderately low (43% of the control mean) with normal Km for 4-methylumbelliferyl alpha-D-glucoside. The hydrolysis of glycogen was markedly decreased (14% of the control mean), and the Km for maltose was increased 4-fold and for glycogen 5-fold. The biosynthesis and the posttranslational processing of the mutant enzyme appeared normal, but the total amount of the enzyme was lower than normal. This mutant enzyme comigrated with normal acid alpha-glucosidase on starch gel electrophoresis, and not with the rare isozyme, acid alpha-glucosidase 2. A possible role of this mutant enzyme in the pathogenesis of this disease and the relationship to glycogenesis II are discussed.

Severe course of glycogen storage disease type II (Pompe's Disease) without development of cardiomegalia

Glycogen storage disease type II Pompe (GSD II) is a lysosomal storage disease caused by an inherited deficiency of acid alpha-glucosidase. In addition to the classical infantile form of GSD II, several clinical variants are known. We describe an infant with the classical course of the disease. Our patient differs from the classical variant by the lack of cardiomegalia and the high residual activity of acid alpha-glucosidase in cultivated skin fibroblasts and muscle tissue. In the present case, however, glycogen storing lysosomes were found in peripheral lymphocytes and skeletal muscle cells. This finding underlines the particular value of ultrastructural investigation in the diagnosis of GSD II.

Developmental study of alpha-glucosidases in Japanese quails with acid maltase deficiency.

In Japanese quails with late-onset acid maltase deficiency (AMD), the activity of acid alpha-glucosidase was severely reduced to approximately 16% of the normal level from an embryonic age. The kinetic characteristics and inhibition by Zn indicated that the residual activity was responsible for the intrinsic activity of acid alpha-glucosidase. However, in affected embryos, the glycogen content and other lysosomal enzyme activities were normal, despite the low acid alpha-glucosidase activity. In a separate study, we found the existence of two age-dependent neutral alpha-glucosidases--"embryonic" and "adult" alpha-glucosidases. In affected quails, the transition from the embryonic neutral alpha-glucosidase to the adult type was not influenced by the disease. The activity toward maltose and glycogen of the embryonic neutral alpha-glucosidase may explain the normal glycogen content in the affected embryos.

Multiple implantation of normal amnion into cattle with generalized glycogenosis type II.

One normal and two carrier calves, and two calves affected by generalized glycogenosis type II were given multiple transplants of normal bovine amnion inserted below the external oblique abdominal muscle in the flank. The interval between transplants was approximately 5 months. The amniotic tissue was not acutely rejected but a host versus graft reaction did occur. The level of acid alpha-glucosidase activity in the amniotic tissue fell rapidly but was still present 2 months after transplantation. Enzyme levels in blood or other tissues did not rise. Excess glycogen deposition and muscle damage occurred in the affected calves and appeared to progress at a rate similar to that in untreated affected animals. One of the affected animals died aged 16 months, which was the same age as the longest surviving non-treated animal in our herd. The other treated animal died aged 24 months.

Isolation and characterization of three alpha-glucosidases from the Japanese quail.

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.

Major contribution of epididymis to alpha-glucosidase content of ram seminal plasma.

Acid alpha-glucosidase and L-carnitine (a well-known epididymal marker) were measured in rete testis and epididymal fluids of adult male rams. These fluids were collected by selective catheterization or by a micropuncture technique, respectively. Both parameters remained at a low and constant level in rete testis and all along caput and corpus epididymidis. Then they increased at equivalent rates in cauda epididymidis to much higher levels than those in seminal plasma (5 mU/mg protein and 10 mM, respectively). An optimum pH study of alpha-glucosidase activity in these fluids showed two well-separated peaks in rete testis and caput epididymal fluids around pH 4 and 7, respectively, but only a single peak at pH 4 in cauda epididymidis that was comparable to the one in seminal plasma. Sucrose density gradient fractions analyzed for their enzyme content in the absence or presence of sodium dodecyl sulfate (1% w/v), a selective inhibitor of acid alpha-glucosidase activity, allowed the demonstration of two enzyme forms at pH 6.8 in rete testis fluid sedimenting in the 7S and 4S regions of the gradient, while a unique 4S form was encountered in cauda epididymidis and in seminal plasma. Although the fate of the minor 7S component of the rete testis fluid in its epididymal transit is presently unknown, similarities between the enzyme in cauda epididymidis and seminal plasma are strong enough to support the hypothesis that epididymis contributes primarily to the acid alpha-glucosidase content of ram seminal plasma.

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull.

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid alpha-glucosidase. The activity of neutral alpha-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity. After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid alpha-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-glucosidase ranged from 3.75 to 4.5 and that of the neutral enzyme from 6.5 to 7.0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.

Heterozygote detection in a family of Lapland dogs with a recessively inherited metabolic disease: canine glycogen storage disease type II

The control of recessively inherited inborn errors of metabolism may benefit from quantitative biochemical screening assays enabling the identification of heterozygous individuals. Based on the principle of partial enzyme deficiency in heterozygotes, an attempt was made to identify heterozygous animals in a Lapland dog family with canine glycogen storage disease type II (acid alpha-glucosidase deficiency). Acid alpha-glucosidase activity was determined in peripheral blood leucocyte extracts of 12 related Lapland dogs, two of which were obligate heterozygotes. The use of an antiserum against acid alpha-glucosidase was necessary to increase the specificity of the assay. Twice the obligate heterozygous enzyme level was assumed to indicate the homozygous normal level. Five dogs were designated as presumptive heterozygotes, and five as presumptive normal homozygotes. The results in two dogs were inconclusive. The information obtained in this preliminary investigation may be helpful in the control of the disease in the Lapland dog breed.