Purification and characterization of a microsomal bile acid beta-glucosidase from human liver.

A human liver microsomal beta-glucosidase has been purified to apparent homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis where a single protein band of Mr 100,000 was obtained under reducing conditions. The enzyme was enriched about 73, 000-fold over starting microsomal membranes by polyethylene glycol fractionation, anion exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by affinity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhibition of activity. The enzyme catalyzed the hydrolysis of 3beta-D-glucosido-lithocholic and 3beta-D-glucosido-chenodeoxycholic acids with high affinity (Km, 1.7 and 6.2 microM, respectively) and of the beta-D-glucoside (Km, 210 microM) and the beta-D-galactoside of 4-methylumbelliferone. The ratio of relative reaction rates for these substrates was about 6:3:11:1. No activity was detectable toward 6beta-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta-D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitation studies using antibodies prepared against lysosomal glucocerebrosidase showed no cross-reactivity with microsomal beta-glucosidase suggesting that these two enzymes are antigenically unrelated.

N1-alkyl-D-gluconamidines: are they 'perfect' mimics of the first transition state of glucosidase action?

The inhibition of four beta-glucosidases of plant, fungal, and mammalian origin by N1-butyl- and N1-dodecyl-D-gluconamidine was determined. Comparison with the inhibition by the corresponding N-alkyl-D-glucosylamines revealed that the strongly basic amidines (pKa 10.8) were at the most 10-times more inhibitory than the weakly basic glucosylamines (pKa 6.5). The small enhancement of inhibitory potency, resulting from transforming the tetrahedral C-1 geometry of the glucosylamines to the planar sp2-geometry of the amidines, was ascribed to the inability of the fully protonated amidines to function as hydrogen bond acceptors with the catalytic acid of the enzyme. Additional evidence for the importance of a hydrogen bond for strong inhibition came from the comparison of K1-values of the weakly basic 5-amino-5-deoxyhexopyranoses and 1,5-iminohexitols with those of the corresponding glyconamidrazones (pKa 8.4), which also have a planar C-1 geometry but are largely protonated under the assay conditions and which had similar or up to 10(4)-times larger K1-values than the former. Transition state resemblance was judged from the ratio KS(alkyl beta-glucoside)/K1(alkyl gluconamidine) relative to the rate acceleration factor kcat/kuncat (Wolfenden, Acc, Chem. Res., 5 (1972) 10-16). Compared to ratios of kcat/kuncat from > or = 10(11) to > or = 10(13), the ratios for KS/K1 were only from 10(3) to 2 x 10(4) except for beta-glucosidase A3 from Asp. wentii which had KS/K1 2.8 x 10(6). This enzyme differs from the others by being strongly inhibited by cationic glycon and substrate analogues rather than by basic ones. The pH-dependence of 1/K1 and the 'slow' approach to the inhibition is discussed with respect to transition state resemblance.

A novel approach to the 1-deoxynojirimycin system: synthesis from sucrose of 2-acetamido-1, 2-dideoxynojirimycin, as well as some 2-N-modified derivatives.

6-Azido-1,3,4-tri-O-benzyl-6-deoxy-D-fructofuranose can be easily obtained in two steps from the known 6,6'-diazido-6,6'-dideoxysucrose (available in two steps from sucrose) and cyclized by controlled hydrogenation and concomitant intramolecular reductive amination to give 3,4,6-tri-O-benzyl-1,5-dideoxy-1,5-imino-D-mannitol, a partially protected derivative of 1-deoxymannojirimycin. After N-protection, position 2 is regio-specifically available to modification. This novel approach was taken advantage of in a synthesis of 2-acetamido-1,2- dideoxynojirimycin and new analogues thereof. Results of inhibition studies conducted with these new compounds employing N-acetylhexosaminidases of various sources are discussed.

[Sugar analog inhibitors for glycosidases, tools for the elucidation of enzymatic hydrolysis of glycosides]. / Zuckeranaloge Inhibitoren für Glykosidasen, Werkzeuge zur Aufklärung der enzymatischen Glykosidspaltung.

Sugar derivatives with a basic group on C-1 (glycosylamines, 5-amino-5-deoxypyranoses, and 1,5-iminohexitols) are bound by most glycosidases 10(2)- to 10(5)-fold more tightly than their nonbasic counterparts. This high affinity and an up to 10(5)-fold better inhibition relative to hexoses by hexono-delta-lactones and lactams point to a catalytic mechanism characterized by a transition state with a partial positive charge and planar geometry at the anomeric carbon of the substrate. Protonation of the glycosidic oxygen atom and stabilization of the positive charge by a carboxylate group strongly shielded from the aqueous environment lower the free energy of activation to an extent which causes an up to 10(14)-fold rate acceleration relative to the nonenzymatic hydrolysis of glycosides.

Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.

The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.

Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.

Bovine N-acetyl-beta-D-glucosaminidase: affinity purification and characterization of its active site with nitrogen containing analogs of N-acetylglucosamine.

Two N-acetylglucosaminidases were isolated from bovine kidney with a three step procedure featuring affinity purification on 2-acetamido-1,2,5-trideoxy-1,5-iminoglucitol (2-acetamido-1,2-dideoxynojirimycin, II). The major isoenzyme, Hex A, is an alpha, beta hetero-dimer (57 and 52 kDa) with isoelectric points from pH 5.3 to 6.6 and comprised about 80% of the total activity. Its kinetic properties with respect to discrimination between N-acetylglucosaminide, N-acetylgalactosaminide and the corresponding 6-sulfate ester were similar to human hexosaminidase A. The minor isoenzyme, Hex B, a homodimer, isoelectric points 7.0 to 7.4, was similar to Hex A but was without detectable activity with methylumbelliferyl-N-acetyl-beta-glucosaminide-6-sulfate. Inhibition studies with Hex A were carried out with 2-acetamido-2,5-dideoxy-1,5-imino-D-glucopyranose (2-acetamido-2-deoxynojirimycin, (1), the corresponding 1,5-lactam (III), with II and its N,N-dimethyl derivative, and with 2-acetamido-2-deoxy-D-glucono-1,5-lactone (IV). In comparison with N-acetylglucosamine (Ki 1.9 mM) Hex A was inhibited 10(6)-fold better by I, 2600-fold better by II, 2900-fold better by III, and 55,000-fold better by IV. A slow approach to the inhibition equilibrium was observed with I and IV. For IV and Hex A it is the first example of a slow inhibition of a glycoside hydrolase by the corresponding glycono-1,5-lactone. The pH-dependence of Ki for the permanently cationic N,N-dimethyl II (15.4 microM (pH 3.5) to 0.47 microM (pH 7.0)) indicated that formation of the enzyme inhibitor complex is governed by deprotonation of a group with pKa 5.0. The results are discussed with respect to structural features and water accessibility of the active site.

Intracellular activity of lysosomal glucosylceramidase measured with 4-nonylumbelliferyl beta-glucoside.

Enzymatic activity of lysosomal glucosyl-ceramidase was determined in intact murine hybridoma and macrophage cells with the synthetic substrate nonylumbeliferyl-beta-glucoside (NUG). The substrate was applied as complex with bovine serum albumin (two binding sites, Kd 2.2 +/- 0.3 microM). The transport of the artificial substrate from medium to the enzyme was explored by measurements of substrate concentrations in cellular membranes and of endocytosis rate relative to substrate hydrolysis. The results indicated that, after enrichment in the plasma membrane, the substrate is mainly transported by simple diffusion. Release of nonylumberlliferone monitored fluorimetrically after disintegration of the cells in borate buffer containing Triton X-100 at pH 9.5 showed that 10(8) cells of both cell lines hydrolysed 1-1.5 nmol substrate/min at a total concentration of 0.1 mM NUG in the medium. Substrate hydrolysis was prevented by preincubating the cells with conduritol B epoxide (CBE), a specific active site-directed inhibitor of lysosomal glucosylceramidase. The substrate concentration at the site of the enzyme and maximal activity were evaluated by the inhibiting effect of the substrate on the inactivation rate by conduritol B epoxide. The rate of inhibitor uptake measured with bromo-[3H]conduritol B epoxide was shown to be not rate-limiting for the inactivation reaction. The molar concentration of the enzyme was determined by labeling with bromo-[3H]conduritol B epoxide. Comparison of the maximal intracellular activity with that of the enzyme after disintegration and activation by taurocholate showed a 20-fold lower activity in the native environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites.

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.

Study of beta-glucosidase from Helix pomatia by active site-directed inhibitors.

This work describes the purification of a beta-glucosidase (beta-D-glucoside-glucohydrolase EC 3.2.1.21) from the digestive juice of Helix pomatia and the study of the enzyme's active site by using different reversible and irreversible inhibitors. The catalytic constants of arylglycosides and their pH-dependent variations have also been determined. The inhibition studies demonstrate that conduritol epoxides are irreversible inhibitors of beta-glucosidase from the digestive juice of H. pomatia, and that nojirimicin shows tight binding with glucosidase: the formation and dissociation of the enzyme-inhibitor complex (dissociation constant 1.1 mumol/1) required several minutes.

Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives.

A beta-glucosidase/beta-galactosidase with Mr 52,500 was isolated from calf liver cytosol by a four-step procedure incorporating affinity chromatography on N-(9-carboxynonyl)-deoxynojirimycin-AH-Sepharose. Its pH optimum was at 5.8 with half-maximal activity at pH 3.5 and 8.6. Affinity for gluco compounds expressed by Km or Ki of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower Vmax than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for kcat/Km of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with Ki (= Km) 2.2 microM and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl beta-glucoside. The product sphingosine was inhibitory with Ki 0.30 microM. A systematic study with alkyl glucosides and glucosylamines defined the aglycon site as a narrow, strongly hydrophobic cleft able to accommodate up to 10 methylene groups. Each CH2 group contributed 3.1 kJ/mol to the standard free energy of binding. The inhibition by gluco- and galactosylamine and by 1-deoxynojirimycin and its D-galacto analog was approximately 200-fold better than by corresponding nonbasic compounds. pH dependence of the inhibition and comparison with permanently cationic glycosyl derivatives showed that the nonprotonated form was the inhibiting species. This feature puts the cytosolic beta-glucosidase in the large class of glycoside hydrolases which strongly bind basic glycosyl derivatives by their protonation at the active site and formation of a shielded ion pair with the carboxylate of an aspartic or glutamic side chain.

Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F.

Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.

Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites.

Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of alkyl(Cn; n = 0-18)amines, alkyl beta-glucosides and alkyl-1-deoxynojirimycins were a biphasic function of increasing chain length: i.e., large decreases in Ki,app or IC50 were found only with n greater than 4 and limiting values were approached with n = 12-14. This biphasic function of alkyl chain length was observed in the presence or absence of detergents and/or negatively charged lipids. In the presence of Triton X-100 concentrations greater than the critical micellar concentration, the relative (to deoxynojirimycin) inhibitory potencies of the N-Cn-deoxynojirimycins (n greater than 4) were decreased about 3-5-fold, due to an energy requirement to extract the inhibitors from Triton X-100 micelles. The Ki,app or IC50 of N-hexylglucosylsphingosine was inversely related to the Triton X-100 concentration and was not affected by the presence of 'co-glucosidase'. The mutual exclusion of glucon, N-Cn-deoxynojirimycin and sphingosine derivatives from the normal enzyme suggested a shared region for binding in the active site. Increasing the fatty-acid acyl chain length of glucosyl ceramide from 1 to 24 carbons had minor effects on Km,app ( = Kis,app) (8-40 microM), but increased Vmax,app up to 13-fold. With the AJGD enzyme, the inhibitor and alternative substrate findings were similar to those with the normal enzyme, except that Kis,app(AJGD)/Kis,app(normal) = 4 to 11 for the Cn-glycons and sphingosine derivatives. These results indicated that (1) the Ki,app or Km,app values for amphiphilic inhibitors or substrates reflect a balance of binding energies for two hydrophobic subsites within the enzyme's active site and Triton X-100 micelles and (2) the abnormal properties of the AJGD enzyme result from an amino-acid alteration(s) within or near a hydrophilic region which is shared by the glycon-binding site and the two hydrophobic sites of the active site.

Further studies on the catalytic mechanism of human liver alpha-L-fucosidase.

Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypothetical fucosyl oxocarbonium ion intermediate.

Partial purification and characterization of Naegleria fowleri beta-glucosidase.

Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction. The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S. The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.

The role of N-glycosylation for the plasma clearance of rat liver secretory glycoproteins.

The clearance of total rat liver secretory glycoproteins and of alpha 1-acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N-glycosylation. Tunicamycin was used for the synthesis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1-deoxymannojirimycin for the synthesis of high-mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid-type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high-mannose-type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid-type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated alpha 1-acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half-lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high-mannose type less than 1 min. In the recirculating perfused liver 28% of complex-type alpha 1-acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high-mannose-type alpha 1-acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N-glycosylation and processing to complex-type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.

Synthesis of 5-amino-5-deoxy-D-galactopyranose and 1,5-dideoxy-1,5-imino-D-galactitol, and their inhibition of alpha- and beta-D-galactosidases.

A 12-step route is presented starting from 1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose for the preparation of the title compounds and their L-altro analogues. Their synthesis is based on the reduction with Raney nickel of a protected 5-hydroxyimino derivative of L-arabino-hexofuranos-5-ulose, with the following improvements for the preparation of a D-galactofuranose derivative: oxidation at C-3 with pyridinium dichromate-acetic anhydride, stereospecific reduction of a 3-O-acetyl-hex-3-enofuranose intermediate to the D-gulo derivative, and inversion at C-3 of its 3-tosylate with tetrabutylammonium acetate in chlorobenzene. alpha-D-Galactosidase from coffee beans and from Escherichia coli and beta-D-galactosidase from E. coli and Aspergillus wentii were inhibited with Ki values that ranged from 0.0007 to 8.2 microM. Formation of the enzyme-inhibitor complexes with the D-galactose analogue was on the time-scale of minutes, whereas the D-galactitol analogue showed a slow approach to the inhibition only with alpha-D-galactosidase from coffee beans and beta-D-galactosidase from A. wentii. N-Alkylation of the D-galactitol analogue was detrimental to the inhibition except for beta-D-galactosidase from E. coli and beta-D-glucosidase from almonds, but, even with these enzymes, the observed affinity enhancements were 10(2) to 10(3)-times smaller than those of N-alkylated D-galactosylamine and D-glucosylamine.