Arnold L. Miller

Electrophoretic forms of human liver α-l-fucosidase and their relationship to fucosidosis (mucopolysaccharidosis F)

Abstract 1. Human liver α-L-fucosidase was studied by starch gel electrophoresis and isoelectric focusing both before and after treatment with neuraminidase. These studies revealed the presence of 5–6 bands on starch gels and six isoelectric forms with p I s of 6.9, 6.5, 6.1, 5.9, 5.7 and 5.5. The activity of the two most acidic forms was reduced after neuraminidase treatment. 2. Liver tissue from a patient with fucosidosis had approximately 4% of normal α- l -fucosidase activity. Starch gel electrophoresis revealed that all electrophoretic forms of the enzyme appear to be deficient. 3. Apparent Michaelis constants ( K m s) determined for both the normal and fucosidotic liver α- l -fucosidase for 4-methylumbelliferyl-α- l -fucopyranoside were found to be 0.085 mM and 0.058 mM, respectively. 4. The pH activity profile of normal and fucosidotic liver α- l -fucosidase were very similar with major pH optima at 5.4 and 5.3, respectively.

Rat liver alkaline phosphatases. Evidence hepatocyte and portal triad enzymes differ.

Using biochemical and electron microscopic histochemical techniques, we studied membrane-bound alkaline phosphatase activities of rat hepatocytes and portal triads. Activity in portal triads was localized to capillaries surrounding bile ducts (peribiliary plexus) and arterioles. Despite the reputation of alkaline phosphatase as a “biliary enzyme,” activity was not observed in bile ducts. Livers were separated into hepatocyte and portal triad fractions with collagenase. Enzyme from hepatocytes migrated faster during electrophoresis and eluted later during anion-exchange chromatography than that from portal triads. Thus, hepatocyte enzyme is more negatively charged (and also possibly smaller) than portal triad enzyme. Twelve hours after bile duct obstruction, new activity appeared on lateral and sinusoidal membranes of hepatocytes; appearance of portal triads did not change with obstruction. Electrophoretic mobilities of the two forms were not altered by obstruction. We conclude that two distinct liver alkaline phosphatases exist, one in hepatocytes, the other in portal triad blood vessels.

Purification of human liver acid β-d-galactosidases using affinity chromatography

Abstract Affinity column chromatography was utilized to purify human liver acid β- d -galactosidases (EC 3.2.1.23). A 21,720 and a 21,951-fold purification was obtained with respect to 4-methylumbelliferyl β- d -galactoside and GM 1 ganglioside, respectively, employing a sequence of concanavalin A-Sepharose and Sepharose 4B-6-aminohexyl 1-thio-β- d -galactopyranoside column chromatography. This acid β- d -galactosidase preparation represents the highest specific activities yet reported for the human liver enzymes. Starch-gel electrophoresis followed by a slicing zymogram indicated the presence of both acid β- d -galactosidase A and B activities, whereas acrylamide-gel electrophoresis at a basic pH demonstrated one band of protein coincident with one peak of enzymatic activity.

Mod A: A post-translational mutation affecting phosphorylated and sulfated glycopeptides in Dictyostelium discoideum

SummaryThe Mod A mutation inDictyostelium discoideum results in a post-translational modification which reduces the activity and electrophoretic mobility of a group of lysosomal glycoproteins. To determine whether this mutation might affect protein bound oligosaccharides, metabolically labeled [2]3H-mannose glycopeptides were isolated from wild-type (AX3) and mutant cells (M31) ofDictyostelium discoideum. A group of large, negatively charged glycopeptides are significantly depleted in strain M31 compared to AX3. Cells of each strain double labeled with3H-mannose and35SO4 or32PO4 showed that the large, negatively charged glycopeptides of AX3 contain both sulfate and phosphate while those of M31 are depleted in these groups. The kinetics of35SO4 release from the glycopeptides of each strain suggested that both contained similar sulfated sugar(s), but that M31 glycopeptides contained three-fold less than those of AX3. Acid hydrolysis of32PO4 containing3H-mannose glycopeptides showed the presence of3H-mannose-6-32-P-phosphate in the AX3 hydrolysates while the glycopeptides of M31 contain only 15% as much mannose-6-phosphate as those of AX3.

Ganglioside GM2 N-acetyl-ß-D-gafactosaminidase and asialo GM2 (GA2) N-acetyl-ß-D-galactosaminidase; studies in human skin fibroblasts

Ganglioside GM2 and its asialo‐derivative, GA2 were radiolabeled in their N‐acetyl‐D‐galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 × 104 dpm/nmol (GM2) and 1.8 × 106 dpm/nmol (GA2) were achieved. About 98% of the label was in N‐acetyl‐D‐galactosamine. Using these substrates, an assay was developed for GM2‐N‐acetyl‐β‐D‐galactosaminidase (E.C.3.2.1.30) and GA2‐N‐acetyl‐β‐D‐galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the GM2 cleaving reaction were identified as N‐acetylgalactosamine and ganglioside GM3‐ Both GM2 and GA2 cleaving activities were stimulated about 5‐fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate GM2‐N‐acetyl‐β‐D‐galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside GM2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for GA2‐N‐acetyl‐β‐D‐galactosaminidase and 4700 for 4‐methylumbelliferyl‐N‐acetyl‐β‐D‐glucosaminidase. Supernates from two patients with Tay‐Sachs disease had markedly reduced activity levels for GM2‐N‐acetyl‐β‐D‐galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoffs disease had reduced activities for all three substrates. A supernate from one patient with juvenile GM2 gangliosidosis cleaved GM2 at a somewhat faster rate than those from Tay‐Sachs or Sandhoffs patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU‐N‐acetyl‐β‐D‐glucosaminide as substrate had approximately half‐normal activities using GM2 as substrate. A patient with the Tay‐Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4‐MU substrate had a profound deficiency using GM2 as substrate. In such unusual hexosaminidase mutants, assays using GM2 as substrate are better indicators of phenotype than those using synthetic substrates.

Purification of human placental α-l-fucosidase by affinity chromatography

Abstract Human placental α- l -fucosidase has been purified 670-fold with a 57% yield by a one-step affinity chromatographic procedure using agaroseepsilon-amino-caproyl-fucosamine.

Purification of Ulex europeus hemagglutinin I by affinity chromatography.

Ulex europeus hemagglutinin I has been purified 95-fold to apparent homogeneity by affinity chromatography on a column of Sepharose 4B containing covalently bound l-fucose. The purified hemagglutinin migrated as a single band of protein upon polyacrylamide gel electrophoresis at an acidic or basic pH. A single precipitin band was observed upon double diffusion of the purified hemagglutinin or crude Ulex europeus extract versus antiserum prepared against the purified hemagglutinin.

Lysosomal enzyme activity in human aqueous humor

Activity of the lysosomal enzymes N-acetyl-beta-D-hexosaminidase, alpha-D-mannosidase, beta-D-glucuronidase, and beta-D-galactosidase was detected in aqueous humor from eyes undergoing intraocular surgery. There was no correlation between lysosomal enzyme activity and age. Lysosomal enzyme activity in human released by ocular tissues surrounding the anterior chamber including the cornea, trabecular meshwork, ciliary body, iris, and lens. Their release into aqueous humor may have a role in regulating aqueous outflow in normal and glaucomatous eyes.

I-cell disease and pseudo-Hurler polydystrophy: heterozygote detection and characteristics of the altered N-acetyl-glucosamine-phosphotransferase in genetic variants

The human disorders I-cell disease and pseudo-Hurler polydystrophy (also known as mucolipidosis II and III, respectively) are caused by an inherited deficiency of UDP-GlcNAc: lysosomal enzyme precursor GlcNAc-P transferase activity. The most common genetic variants of these diseases (complementation group A) can be identified in homozygotes and heterozygotes using a GlcNAc-P transferase assay with artificial acceptors and commercially available radiochemicals. The kinetic characteristics of the residual GlcNAc-P transferase activity in complementation group A fibroblasts indicates that the low activity is due to a low Vmax. The measured Michaelis-Menten constants for the substrates UDP-GlcNAc and alpha-methyl mannoside are in the normal range. Homozygotes and heterozygotes of another less common variant of pseudo-Hurler polydystrophy (complementation group C) have normal activity and normal kinetic characteristics with this assay using alpha-methyl mannoside as the acceptor substrate. Several PHP variants with unusual characteristics are discussed.

Altered serum α-D-mannosidase activity in mucolipidosis II and mucolipidosis III

Abstract Normal human serum contains at least three forms of α-D-mannosidase: an acidic form which has a pH optimum of 4.25, is inhibited by Co2+ and is thermostable; an intermediate form, which has a pH optimum of 5.6–5.7, is stimulated by Co2+ and is heat labile at 50°C; and a neutral form with a pH optimum of 6.0–6.5. In Mucolipidosis II and III sera, the acidic α-mannosidase activity persists while the intermediate activity is absent or altered. Heating the serum does not affect the pH activity curve, the electrofocusing profile or the response to Co2+ of α-mannosidase. During heat inactivation at 55°, 90–100% of the pH 5.6 α-mannosidase activity is lost in normal sera while less than 40% is lost from ML sera. The effect on sera from ML obligate heterozygotes is intermediate. The absent or altered intermediate mannosidase may be responsible for aberrant biochemical properties reported for other glycosidases in Mucolipidosis II and Mucolipidosis III.