Elizabeth F. Neufeld

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? Evidence from I-cell disease.

Abstract We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β32P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α- L -iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.

Formation and interconversion of sugar nucleotides by plant extracts

Abstract Extracts from mung bean seedlings and from a number of other plants contain a pyrophosphorylase (or pyrophosphorylases) capable of catalyzing the reversible formation of sugar nucleotides from UTP and a number of sugar 1-phosphates, according to the following reaction: UTP + S-1-P UDPS + PP. UDPG, UDPGal, UDPXy, and UDPAr are formed in this reaction from α- d -G-1-P, α- d -Gal-1-P, α- d -Xy-1-P, and α- and β- l -Ar-1-P, respectively. The reaction requires a bivalent metal ion (Mg ++ , Mn ++ , or CO ++ ). Sugar nucleotides are not formed from β- d -G-1-P, β- d -Gal-1-P, β- d -Xy-1-P, α- d -Ar-1-P, α-, β- l -Ar(F)-1-P, or α- d -Ar(F)-1-P. No exchange between PP and UTP 32 can be demonstrated except in the presence of one of the reactive sugar 1-phosphates. Mung bean seedling extracts also catalyze the interconversion of UDPG and UDPGal and of UDPXy and UDPAr.

Formation of galactolipids by chloroplasts.

Chloroplasts of higher plants contain relatively large amounts of galactolipids (Wintermans, 1960). These have been shown to be α, β diglycerides (predominantly dilinolenin), with either one or two D-galactosyl residues attached to the third hydroxyl group of glycerol (Benson et, al., 1958; Carter et, al., 1961; Sastry and Kates, 1963a). In the present investigation it was found that isolated spinach chloroplasts catalyze the transfer of galactose from UDP-D-galactose-C14 to an endogenous acceptor, yielding alkali-labile products which are similar, though not identical, to the galactolipids isolated from plant material.

Biosynthesis of Saccharides from Glycopyranosyl Esters of Nucleotides (“Sugar Nucleotides”)

Publisher Summary This chapter discusses the enzymic synthesis of numerous glycosides, including oligosaccharides and polysaccharides, which is subsequently effected by the transfer of a glycosyl residue from a saccharide donor to an appropriate acceptor. Transglycosylations involve only a redistribution of glycosidic linkages among saccharides, not a net increase in the number of such linkages. Another mechanism for the enzymic synthesis of saccharides is one that occurs through the transfer of the glycosyl group from a glycosyl phosphate to an appropriate acceptor. The evidence for the participation of glycosyl nucleotides in the biosynthesis of numerous glycosides, including disaccharides and polysaccharides, are described in the chapter.

Enzymic synthesis of uridine diphosphate glucuronic acid and uridine diphosphate galacturonic acid with extracts from Phaseolus aureus seedlings.

Abstract Extracts from mung bean seedlings contain a pyrophosphorylase (or pyrophosphorylases) capable of catalyzing the reversible formation of the following compounds: (a) uridine diphosphate d -glucuronic acid and inorganic pyrophosphate from uridine triphosphate and d -glucuronic acid 1-phosphate; and (b) uridine diphosphate d -galacturonic acid and inorganic pyrophosphate from uridine triphosphate and d -galacturonic acid 1-phosphate.

Enzymic phosphorylation of d-glucuronic acid by extracts from seedlings of Phaseolus aureus

Abstract Enzyme preparations capable of phosphorylating d -glucuronic acid in the presence of ATP and MgCl 2 were obtained from mung bean seedlings. Both soluble and particulate preparations catalyzed the reaction. The enzymically formed phosphate ester was identified as α- d -glucuronic acid 1-phosphate.

Characterization of the factor deficient in the Hunter syndrome by polyacrylamide gel electrophoresis

Abstract Fibroblasts cultured from skin of patients with the Hunter syndrome lack a genotype-specific macromolecular factor required for mucopolysaccharide degradation. From its electrophoretic mobility at several gel concentrations, this factor was estimated to have a molecular weight of 65,000 and a valence of −11 at pH 8. We conclude from these data that the factor is a protein.

Hydrolysis of amylose by β-amylase and Z-enzyme

Abstract 1. 1. The observation of Peat et al. (7) that potato amylose is incompletely hydrolyzed with pure β-amylase unless another factor (Z-enzyme) is added has been confirmed. 2. 2. Treatment with hot alkali of the resistant limit dextrin, obtained after hydrolysis of the amylose with β-amylase, has an effect similar to treatment with Z-enzyme in that it causes the residual dextrin to be further hydrolyzed with β-amylase. 3. 3. The exact degree of β-amylolysis depends on the plant material from which the sample of amylose is derived, on the particular subfraction used, or on the previous treatment of the amylose sample. Like natural amylose, synthetic amylose obtained by the action of potato phosphorylase on α- d -glucose 1-phosphate is not completely hydrolyzed by β-amylase. However, its limit of hydrolysis is considerably greater (89%, increased to 93% by the addition of emulsin). 4. 4. The Z-enzyme activity could be removed from the β-glucosidase and laminarase activities in almonds by purification, indicating that the obstacle to hydrolysis by β-amylase is probably not a β-glucosidic linkage. 5. 5. β-Glucosidase and laminarase are shown to be distinct from each other, as well as from Z-enzyme. 6. 6. The intrinsic viscosity of the limit dextrin is found to be approximately 11% lower than that of the parent amylose. This fact has been discussed with regard to the probable location of the anomalous linkage.

Hydrolysis of glycopyranosyl phosphates of the β-d (or α-l) configuration by a plant phosphatase

Abstract An acid phosphatase hydrolyzing β- d -galactose 1-phosphate has been purified 12-fold from Phaseolus aureus seedlings. It is not affected by Mg ++ but is inhibited by fluoride, molybdate, inorganic phosphate, and some heavy-metal ions. A number of other glycopyranosyl phosphates of the β- d (or α- l ) configuration were also effective substrates, while the α- d (or β- l ) anomers were hydrolyzed at one-twentieth to one-thirtieth the rate. Non-glycosyl phosphates were hydrolyzed at a rate intermediate between that of the α and that of the β anomer, probably as a result of contamination by nonspecific phosphomonoesterase.