Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.     

Partial purification and characterization of an ovarian tripeptidyl peptidase: a lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets

The pregnant hog ovary is a rich source of a novel exopeptidase that catalyzes the release, at pH 5.0, of collagen-related (P3-P2-P1) "triplets" such as Gly-Pro-Met, Gly-Pro-Arg, and Gly-Pro-Ala from arylamide derivatives, provided the N termini are unsubstituted. Corresponding derivatives of related (P2-P1) dipeptides (Pro-Met, Pro-Ala) or (P1) amino acids (Met, Arg, Ala) are not attacked. The enzyme was purified 58-fold from a detergent extract of a water-extracted tissue residue. Activity was determined on Gly-Pro-Met-2-naphthylamide at pH 4.5 and 37 degrees C (Km 0.45 mM; Vmax 722 nmoles/min/mg protein). The responsible Mr 55,000 exopeptidase, termed "tripeptidyl peptidase", forms high-Mr aggregates, belongs to the serine catalytic class, and has a lysosomal localization. Gly-Pro-Ala triplets were released sequentially at pH 5.0 from a Mr 14,000 polypeptide, poly(Gly-Pro-Ala-). When this reaction was coupled to that of homologous dipeptidyl peptidase II, the liberated tripeptides were reduced to dipeptides and free amino acids: (Gly-Pro-Ala)n----nGly-Pro-Ala----nGly-Pro + nAla.     

Molecular screening of the major mutations in the ARSA gene in patients with metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is an inherited storage disease caused by deficiency of arylsulfatase A (ARSA). Molecular analysis of the major mutations in the ARSA gene was performed in 10 Ukrainian patients (from 9 families) with MLD. According to the age of onset, late infantile MLD was identified in 3 patients, juvenile MLD in 5 patients, and adult MLD in 2 patients (sibs), respectively. The ARSA activity in the patients was 2-26 nmol/h/mg protein (the normal activity has been established in our laboratory as 111.9 +/- 7.1 nmol/h/mg protein). No correlation between enzyme activity and a clinical course of disease was revealed. The IVS2 + 1 mutation was found at 2 of 20 alleles (in a patient with late infantile form) and the P426L mutation was found at 2 of 20 alleles (in two patients with juvenile form). Thus, the total frequency of these two major mutations in the ARSA gene is 20% in Ukrainian MLD patients.     

Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.     

Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.     

Beta-glucosidase from Penicillium purpurogenum: purification and properties.

beta-Glucosidase was purified from the culture supernatant of Penicillium purpurogenum. The purified enzyme was homogeneous on both nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme is a monomeric glycoprotein with M(r) of 90,000 as determined by gel filtration on Bio-Gel P-300 and SDS-polyacrylamide gels. Two enzyme forms were resolved by chromatofocusing and isoelectric focusing, and the pI values obtained with both methods were 4.2 (major form) and 6.0. The major form was characterised further. Enzyme activity was optimal at pH 3.5 and at 60 degrees C. The enzyme was stable in the pH range 2.5-9.5 for 24 h at 4 degrees C. Kinetic analysis gave Kms of 0.8 mM for cellobiose and 85 microM for p-nitrophenyl-beta-D-glucopyranoside. The enzyme hydrolyses a wide range of substrates including aryl-beta-glucosides, cellobiose, and amygdalin. Glucose inhibits competitively and glucono-delta-lactone is a mixed inhibitor of the enzyme.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.     

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.     

pH dependence of the reduction of dioxygen to water by cytochrome c oxidase. 1. The P(R) state is a pH-dependent mixture of three intermediates,A, P,and F

Recent studies on cytochrome oxidase have indicated that the putative "peroxy" intermediate in the catalytic cycle (P(R)) is a mixture of intermediates, including P and F [Sucheta, A., et al. (1998) Biochemistry 37, 17905-17914], and the bench-made P and F forms appear to have the same redox state (Fe(a3)(4+)=O(2-)), but a different protonation state [Fabian, M., and Palmer, G. (2001) Biochemistry 40, 1867-1874]. To explore the possibility that the putative P(R) state is a pH-dependent mixture of intermediates, we investigated the reduction of dioxygen to water by the fully reduced cytochrome oxidase at pH 6.2, 7.5, and 8.5 in the visible and Soret regions (350-800 nm) using the CO flow-flash technique. Singular value decomposition and global exponential fitting of the time-resolved absorption difference spectra resolved five apparent lifetimes. The fastest three (1.5, 13, and 34 micros) were independent of pH, while the two slowest rates (80-240 micros and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. When the time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reduced enzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However, the putative P(R) intermediate was best represented by a pH-dependent mixture of compound A, P, and F. The ferryl form was favored at low pH. The subsequent intermediate is a ferryl with a pH-dependent electron transfer equilibrium between heme a and Cu(A), the reduced heme a being favored at low pH. These results suggest a pH-dependent reaction mechanism of the reduction of dioxygen to water by the fully reduced enzyme that is more complex than previously proposed.     

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.     

Biomembrane-modulated, lysosomal phospholipase A2 contamination of chromaffin granule ghosts

It was reported that subcellular fractionation of bovine adrenal medulla results in the separation of distinct, non-calcium-dependent phospholipases A2--one associated with chromaffin granule ghosts, another with lysosomes. The basis of this distinction is pH optimum: in routine assays utilizing neat liposomal substrates, the chromaffin granule ghost-associated enzyme is alkaline-active whereas the lysosomal enzyme is acid-active (Husebye, E.S. and Flatmark, T. (1987) Biochim. Biophys. Acta 920, 120-130). We now report that biomembranes after liposomal substrates and/or lysosomal phospholipase A2 such that the enzyme now hydrolyzes them (at low cation concentration) with an alkaline pH optimum. In a lysosomal membrane fraction, phospholipase A2 activity at pH 7.5 relative to activity at pH 5.0 increases as increasing amounts of lysosomal membranes are assayed. The pH optimum of chromaffin granule ghost-associated phospholipase A2 toward liposomal substrates is likewise biomembrane-dependent and, when assayed carefully, is indistinguishable on the basis of optimal pH from the lysosomal enzyme. Although chromaffin granule ghost-associated phospholipase A2 is most likely a lysosomal contaminant, its broad, biomembrane-modulated pH range may still allow it to participate in catecholamine secretion. More importantly, however, sensitivity of adrenal medullary lysosomal phospholipase A2 to biomembranes broadens its potential physiologic pH range and may also play a role in the regulation of this potentially deleterious activity.     

Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase: a structural analysis of dual NAD(P)H specificity

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N. I. , Parsonage, D., Paige, C., Newton, G. L., Fahey, R. C., Leonardi, R., Jackowski, S., Mallett, T. C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 A resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 A resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367,425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.     

Cofactor-dependent assembly of the flavoenzyme vanillyl-alcohol oxidase

The oligomerization of the flavoprotein vanillyl-alcohol oxidase (VAO) and its site-directed mutant H61T was studied by mass spectrometry. Native VAO has a covalently bound FAD and forms primarily octameric assemblies of 507 kDa. H61T is purified as a FAD-free apoprotein and mainly exists as a dimeric species of 126 kDa. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization, although association of H61T dimers seems not to be crucial for enzyme activity. Reconstitution of H61T with the cofactor analog 5'-ADP also promotes octamerization. FMN on the other hand, interacts with apoH61T without stimulating dimer association. These results are in line with observations made for several other flavoenzymes, which contain a Rossmann fold. Members of the VAO flavoprotein family do not contain a Rossmann fold but do share two conserved loops that are responsible for binding the pyrophosphate moiety of FAD. Therefore, the observed FAD-induced oligomerization might be general for this family. We speculate that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO species are transmitted to the protein surface, promoting oligomerization.     

Proteolysis of isolated mitochondria by myocardial lysosomal enzymes.

1. Solubilized mitochondria and lysosomal fractions were obtained from guinea-pig heart by differential centrifugation and selective membrane disruption. 2. Mitochondria incubated at 37 degrees C in the presence of lysosomal enzymes underwent proteolysis. The rate of protein degradation was inversely dependent on pH. 3. The use of proteinase inhibitors showed that at low pH the major enzyme involved in mitochondrial digestion was cathepsin D. 4. At neutral pH carboxyl proteinases were still active, but thiol proteinases accounted for most of the protein breakdown. 5. The role of lysosomal enzymes as mediators of mitochondrial damage in ischaemic myocardium is discussed.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Crystallographic studies on D-amino acid oxidase.

D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.     

Properties of mouse alpha-galactosidase.

alpha-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-alpha-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzmye was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a Km value of 1.4 mM, and energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human alpha-galactosidase A. The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the alpha-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.