Ganglioside GM1 beta-galactosidase: studies in human liver and brain

A microcolumn assay for ganglioside GM₁ β-galactosidase (EC 3.2.1.23) has been developed using GM₁ tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β-d-galactopyranoside is hydrolyzed about seven times more rapidly than GM₁ in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM₁ β-galactosidase in both brain and liver. The major form (ganglioside GM₁ β-galactosidase A) had a molecular weight of 60–70 × 10³ and the minor form (ganglioside GM₁ β-galactosidase B) 600–800 × 10³. The liver and brain GM₁ β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens) suggesting that both are sialylated glycoproteins. The activity of GM₁ β-galactosidase in the brain and liver tissue of patients with GM₁ gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM₁ gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM₁ β-galactosidases.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Complementation studies with enucleated fibroblasts from different variants of beta-galactosidase deficiency.

Somatic cell hybridization of β-galactosidase fibroblasts derived from patients with the infantile type 1 and the adult type 4 G_M1-gangliosidosis results in restoration of the β-galactosidase activity. The kinetic properties of the enzyme activity in heterokaryons were found to be similar as in controls. Genetic complementation did not occur after inhibition of protein synthesis by cycloheximide indicating the necessity of de novo protein synthesis. When fibroblasts of the adult type 4 patient were enucleated and fused with cells from an infantile type 1 variant no restoration of β-galactosidase in the hybrids was observed. Fusion of enucleated type 1 cells with nucleated type 4 cells, however, did result in genetic complementation. The results obtained in this study and observations by others fit with the hypothesis of intergenic complementation. In heterokaryons the adult type 4 genome codes for normal β-galactosidase monomers and the infantile type 1 cells or cytoplasts provide a protein factor necessary for the hydrolytic activity and aggregation of the molecule.     

Neurochemical abnormality in I-cell disease: chemical analysis and a possible importance of beta-galactosidase deficiency.

Sphingolipid composition in both gray and white matter of a patient with I-cell disease was normal except for the higher proportion.of G_MI-ganglioside in gray and white matter. In the patient's liver and kidney there was a significant accumulation of ceramide dihexoside and ceramide trihexoside and of sulphatide in kidney. Non-lipid hexosamine and sialic acid concentration in brain was increased 1.2-1.5 times above normal. Recovery of myelin from I-cell's white matter was 80-100%, suggesting that demyelination, if present, is minimal. Myelin lipid and myelin specific glycoprotein patterns were normal. Except for β-galactosidase activity the activity of other brain lysosomal enzymes were within the normal range. This finding was similar to that of Hurler's syndrome. Only β-galactosidase activity was reduced to less than 10% of normal in the patient's brain. To examine the possible metabolic significance of β-galactosidase deficiency in I-cell disease the physical characteristics of this enzyme, isolated from tissues from I-cell, Hurler and control patients, were compared using isoelectric focusing, Con A-Sepharose and Sephadex G-150 chromatography. The isoelectric point and the binding affinity of I-cell β-galactosidase with Con A-Sepharose was comparable to normal. However, the isoenzyme patterns of brain and liver I-cell β-galactosidase with Sephadex G-150 gel filtration revealed decreased acid β-galactosidase. Effects of the addition of sodium chloride on each fraction of β-galactosidase isoenzymes isolated from I-cell tissues were markedly different from controls, whereas the pH optimum of these enzymes were similar to normal. These enzyme characteristics in I-cell tissues were different from normal and Hurler's syndrome. These findings suggest that β-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than secondary inhibition as found in the mucopolysaccharidoses and thus may have an important role for the pathogenesis of brain damage and disease occurrence.     

Macular cherry-red spots and myoclonus with dementia: coexistent neuraminidase and beta-galactosidase deficiencies.

A patient was previously characterized as having a variant form of G_M1 gangliosidosis based on severe deficiencies in β-galactosidase activity in both leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-galactoside and G_M1 ganglioside. Reexamination of her cultured fibroblasts revealed a severe deficiency in neuraminidase activity using neuramin lactose, fetuin and 2-(3′-methoxyphenyl)-N-acetyl-D-neuraminic acid as substrates, but normal neuraminidase activity using G_M3 ganglioside as a substrate. The presence of normal levels of β-galactosidase activity in leukocytes from the mother of the patient indicates that the β-galactosidase deficiency is not the primary enzyme defect in this type of patient.     

The effect of inflammation on rat liver beta-galactosidase and beta-N-acetylglucosaminidase.

Reduced activities of β-galactosidase and β-N-acetylglucosaminidase were found in rat livers following experimentally induced inflammation. The greatest reduction in glycosidase activities were found 24 h after inflammation. The lysosomal fraction accounted for the bulk of both glycosidase activities in experimental and control rats. Kinetic experiments showed that inflammation did not affect K_m values, but did result in a significant reduction in V_max values. One suggestion to explain the results is that inflammation causes a reduction in biosynthesis or activation of the two glycosidases in rat liver.     

Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The K_eq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S_20,w of 8, while the monomer had an S_20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Purification of acid beta-galactosidase and acid neuraminidase from bovine testis: evidence for an enzyme complex

The isolation of an acid neuraminidase from bovine testis is described. Under all experimental conditions this neuraminidase copurifies with acid β-galactosidase, but not with other lysosomal hydrolases. Immunotitration with an antiserum raised against purified human placental β-galactosidase results in the coprecipitation of both enzyme activities. Our data indicate that acid neuraminidase and β-galactosidase are present as an enzyme complex. The possible physiological relevance is discussed.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Purification and characterization of extracellular β-galactosidase from the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica

A psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica could produce high level (17.2 U/ml) of both extracellular and cell-bound β-galactosidase. The extracellular β-galactosidase in the supernatant of the cell culture of the psychrotolerant yeast G. pullulans 17-1 was purified to homogeneity with a 2.4-fold increase in specific activity as compared to the supernatant by concentration, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-Sepharose Fast Flow cation-exchange). The molecular mass of the purified extracellular β-galactosidase was estimated to be 335 kDa. The optimal temperature and pH of the purified β-galactosidase were 50 °C and 4.0, respectively. K_m and V_max values of the purified β-galactosidase for o-nitrophenyl-β-d-galactopyranoside were 3.3 mM and 9.2 μmol/min. Lactose can be converted into glucose and galactose and a large amount of reducing sugar can be released from milk under catalysis of the purified β-galactosidase. The matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy identified a peptide ALEEYKK which is the conserved motif of the β-galactosidases from other yeasts. The results show that the enzyme may have potential applications in food industry.     

The effect of controlled feeding of glycerol on beta-galactosidase production by Escherichia coli in batch culture

A mutant of E. coli constitutive for β-galactosidase has been grown in batch culture with the carbon source, glycerol, fed at various fixed rates to the culture. High feeding rates where growth was only slightly restricted gave final enzyme levels similar to those obtained in cultures where all the glycerol was added initially. Low feeding rates resulted in breakdown of the β-galactosidase formed and gave reduced final levels of the enzyme.     

Galactosylceramide beta-galactosidase in Krabbe disease: partial purification and characterization of the mutant enzyme

Galactosylceramide β-galactosidase (EC 3.2.1.46) has been partially purified from liver of a patient who died of Krabbe disease. Approximately 700-fold purification was achieved by solubilization, adsorption with immobilized concanavalin A, gel filtration through Bio-Gel A-1.5m and chromatography on immobilized sphingosine. The relative increase in crossreacting material and residual galactosylceramidase and lactosylceramidase I activities of the mutant enzyme was essentially identical to that obtained for the enzyme partially purified by the same procedure from normal liver control. An apparent molecular weight of about 750,000 and similar electrophoretic mobilities were observed for both enzymes. In contrast, catalytic properties and stability of the enzyme protein were severely affected in the mutant as compared to the normal enzyme. The apparent K_m values of the mutant enzyme for β-galactosidase activities toward galactosylceramide and lactosylceramide in the presence of pure sodium taurocholate were 14 and 4 times, respectively, higher than the normal values. Incubation for 4 min at 52 °C or dialysis against 1.3 m urea caused a 50% loss of residual enzymatic activity of the mutant enzyme, whereas a 35-min incubation or dialysis against 5.6 m urea was required for 50% inactivation of the normal enzyme. These findings indicate that the mutation in Krabbe disease leads to synthesis of normal quantities of catalytically and structurally altered protein.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

The effect of oxygen transfer on the activity of encapsulated whole cell β-galactosidase

The effect of oxygen transfer on the production of immobilized whole cell β-galactosidase has been evaluated. The encapsulated whole cell β-galactosidase was prepared by combining cell encapsulation and culture into one-step. Escherichia coli was encapsulated and cultured in the growth and production media to accumulate β-galactosidase in itself. Sunflower seed oil was coimmobilized to increase the oxygen transfer rate through the capsule membrane. The oxygen transfer rate increased 63 percent and the activity of β-galactosidase increased by 10 percent. The activity of encapsulated β-galactosidase obtained in the concentric air lift reactor was 86 percent higher than that in the shaking incubator. In the concentric air lift reactor, the accumulation of encapsulated whole cell β-galactosidase was primarily dependent on the capsule velocity. While the accumulation of specific β-galactosidase in the capsule increased with volumetric oxygen transfer coefficient, the cell biomass accumulated in the capsule decreased.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

Defective phosphorylation and processing of beta-hexosaminidase by intact cultured fibroblasts from patients with mucolipidosis III

Previous studies of the synthesis, phosphorylation, and processing of β-hexosaminidase in cultured fibroblasts from normal individuals and from patients with mucolipidosis II (I-cell disease) (A. Hasilik and E. F. Neufeld, 1980, J. Biol. Chem.225, 4937–4946) have been extended to fibroblasts derived from patients with a related genetic disorder, mucolipidosis III (pseudo-Hurler polydystrophy). The enzyme was biosynthetically labeled in pulse-chase experiments with [³H]leucine and ³³P_i, and isolated from cells and medium by immunoprecipitation. The constitutent α and β chains of the enzyme were separated by polyacrylamide gel electrophoresis under reducing and denaturing conditions, visualized by autoradiography and fluorography, extracted from the gel, and quantitated by liquid scintillation spectrometry. Enzyme produced by fibroblasts from mucolipidosis III patients had a very low but detectable phosphate content; a high proportion of newly made enzyme was secreted, though some remained within the cells and was processed to mature enzyme; the presence of NH₄Cl during the labeling and chase did not significantly increase the amount of enzyme secreted. The β-hexosaminidase produced by mucolipidosis III fibroblasts thus resembled more closely that produced by fibroblasts from patients with mucolipidosis II than the normal enzyme. β-Hexosaminidase made by fibroblasts from mucolipidosis II heterozygotes was similar to the normal enzyme with respect to phosphorylation, processing, and secretion. Mucolipidosis II and III fibroblasts could endocytose normal precursor β-hexosaminidase and process it to the mature form. The deficiency of mature enzyme in the patients' cells may therefore be attributed to failure of the unphosphorylated enzyme to be incorporated into lysosomes, where processing would normally occur.