Biomedical subjects

M L Sinnott

Publications and source records attributed to M L Sinnott.

At least 37 records · Page 2Linked to original sources

PubMed1993

Large changes of transition-state structure during experimental evolution of an enzyme.

The question of whether, during the evolution of an enzyme, the transition state of the catalysed reaction is largely unchanged, or whether transition state and protein change together, was examined using the egb beta-galactosidases of Escherichia coli. Charge development at the first chemical state was assumed [Konstantinidis and Sinnott (1991) Biochem. J. 279, 587-593] to be proportional to delta delta G++, the ratio of second-order rate constants for the hydrolysis of beta-D-galactopyranosyl fluoride and 1-fluoro-D-galactopyranosyl fluoride, expressed as a free-energy difference. delta delta G++ (kJ.mol-1) falls from 10.4 for wild-type enzyme to 6.8 and 7.2 as a consequence of two different single amino-acid changes (which arise from single evolutionary events), to 6.3 as a consequence of the two amino-acid changes together, and then increases slightly to 7.3 as a consequence of a third single evolutionary change involving three further amino-acid changes.

Amino Acid Sequence↗

PubMed1992

The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions.

1. The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions. 2. This datum, taken together with a recalculation of the previous ultracentrifuge data [Hall (1976) J. Mol. Biol. 107, 71-84], analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme. 3. The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol. 4. The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb. 5. The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx. 44 M and that for hydrolysis of lactulose is approx. 40 M. 6. A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles [(1989) Biochemistry 28, 9283-9305] for an enzyme catalysing a thermodynamically irreversible reaction. 7. Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured. 8. The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga. 9. The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.

Biological Evolution↗

PubMed1991

The interaction of 1-fluoro-D-glucopyranosyl fluoride with glucosidases.

1. 1-Fluoro-D-glucopyranosyl fluoride undergoes pH-independent loss of F- ion at a rate of 1 x 10(-8) s-1 at 50.0 degrees C, some 10(3)-fold slower than alpha-D-glucopyranosyl fluoride and 4 x 10(4)-fold slower than beta-D-glucopyranosyl fluoride. 2. The (inverting) amyloglucosidase II of Aspergillus niger hydrolyses the difluoride according to Michaelis-Menten kinetics (Km 34 mM and kcat. 0.27 s-1), by apparently the same (simple) mechanism by which it hydrolyses alpha-D-glucopyranosyl fluoride (Km 38 mM and kcat. 730 s-1), rather than by the Hehre resynthesis-hydrolysis mechanism used to transform beta-D-glucopyranosyl fluoride. 3. The difluoride is also a substrate for the (inverting) trehalase of pig kidney [Km 17.3 mM and Vmax. 6.2 x 10(-4) relative to alpha-D-glucopyranosyl fluoride (Km 38 mM]). 4. The quantitatively similar effect of fluorine substitution on the one-step enzymic reactions and on the non-enzymic reactions suggests that they go through similar (oxocarbonium-ion-like) transition states. 5. The difluoride is a substrate for the (retaining) beta-glucosidases from Aspergillus wentii (A3 enzyme) and sweet-almond meal (B isoenzyme) and for the retaining alpha-glucosidase from rice: comparison with the appropriate monofluoride reveals a variable rate-retarding effect of the second fluorine atom on kcat./Km that correlates with other measures of oxocarbonium ion character in the transition state. 6. The difluoride is a substrate for the (retaining) alpha-glucosidase from yeast, but also gives an insidious mimicry of active-site-directed irreversible inhibition, which we tentatively attribute either to formation of the non-covalent complex or to the fluoroglucosyl-enzyme increasing the well-known tendency of this enzyme to come out of solution by adsorption on the walls of the vessel.

Animals↗

PubMed1991

beta-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction.

1. [2'-2H]Inosine was made from inosine by tetraisopropyldisiloxanyl protection of the 3'- and 5'-positions, oxidation with dimethyl sulphoxide and acetic anhydride, immediate NaB2H4 reduction of the oxo sugar product and inversion at C-2' of the resultant protected [2'-2H]arabino-inosine by trifluoromethanesulphonylation and reaction with caesium propionate, followed by deprotection. 2. The equilibrium-perturbation technique was used to measure beta 2H(V/K) for phosphorolysis of this compound by the purine nucleoside phosphorylase of Escherichia coli as a function of pH. 3. The pH variation indicates an intrinsic effect of 1.068 masked by isotopically silent steps near the pH optimum. 4. The similar pH variation of these beta-deuterium effects and the alpha-deuterium effects measured previously [Stein & Cordes (1981) J. Biol. Chem. 256, 767-772; Lehikoinen, Sinnott & Krenitsky (1989) Biochem. J. 257, 355-359] for this reaction provides the first experimental reassurance for the common assumption that pH changes merely mask and unmask the chemical steps in an enzyme-catalysed reaction, and do not detectably alter transition-state structure. 5. The dihedral angle between the C-H-2' bond and the electron-deficient p-orbital at the transition state is in the range 32-48 degrees, in accord with an essentially planar furanose ring.

Deuterium↗

PubMed1990

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗

PubMed1990

Inhibition of the alpha-L-arabinofuranosidase III of Monilinia fructigena by 1,4-dideoxy-1,4-imino-L-threitol and 1,4-dideoxy-1,4-imino-L-arabinitol.

1. 1,4-Dideoxy-1,4-imino-L-threitol was synthesized and the synthesis of 1,4-dideoxy-1,4-imino-L-arabinitol was improved. 2. Both compounds are competitive inhibitors of Monilinia fructigena alpha-L-arabinofuranosidase III, the additional hydroxymethyl group in the arabinitol contributing about 17.8 kj/mol (4.25 kcal/mol) to the Gibbs free energy of binding. 3. The affinities (1/Ki) of both compounds vary with pH in a classical bell-shaped way, the pKa value being that of the acid-catalytic group on the enzyme [5.9; Selwood & Sinnott (1988) Biochem. J. 254, 899-901] and the pKb values being those of the free inhibitors, 7.6 and 7.8 respectively. 4. On the basis of these and literature data we suggest that efficient inhibition of a glycosidase at its pH optimum by an appropriate iminoalditol will be found when the pKa of the iminoalditol is below that of the acid-catalytic group of the target enzyme.

Arabinose↗

PubMed1989

The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.

Catalysis↗

PubMed1989

Investigation of alpha-deuterium kinetic isotope effects on the purine nucleoside phosphorylase reaction by the equilibrium-perturbation technique.

1. alpha-Deuterium kinetic isotope effects on the phosphorolysis of inosine catalysed by Escherichia coli purine nucleoside phosphorylase were measured by the equilibrium-perturbation technique, by using the change in absorbance at 250 nm (approx. 20%). 2. Values of 2H(V/K) of 1.13(9) at pH 5.0, 1.10(5) at pH 6.1, 1.09(4) at pH 7.3, 1.08 at pH 8.4 and 1.16(4) at pH 9.4 were obtained. 3. These are compared with literature alpha-deuterium kinetic isotope effects for this and related reactions. 4. The equilibrium constant, defined as [inosine].[H2PO4-]/[hypoxanthine] [alpha-Rib f OPO3H-], is 46 at 25 degrees C. 5. N-3-beta-D-Ribofuranosylhypoxanthine, an impurity in chemically synthesized inosine, is a substrate.

Animals↗

PubMed1988

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.

Arabinose↗

PubMed1987

Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena.

1. The alpha-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding & Byrde (1974) J. Gen. Microbiol. 81, 151-163] was purified by sequential isoelectric focusing, hydrophobic chromatography, gel filtration and chromatofocusing. 2. The enzyme is a monomer of Mr 40,000. 3. On inactivation of the enzyme with 3H-labelled 1-alpha-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene, 0.64 mol of alpha-L-arabinofuranosylmethyl residues/mol of enzyme is estimated to become attached to protein. 5. Neither first-order nor second-order rate constants for hydrolyses of aryl alpha-L-arabinofuranosides are dependent upon leaving-group acidity [beta lg(V) = -0.16 +/- 0.11; Beta lg(V/K) = -0.11 +/- 0.07; n = 7; delta pKa = 4.5] 6. Bond-breaking is nonetheless rate-limiting, as is shown by a value of 18(V) of 1.030 +/- 0.007 for the hydrolysis of p-nitrophenyl arabinoside. 7. Proton-donation to the leaving group is thus far advanced at the rate-limiting transition state for this enzyme. 8. Four alpha-L-arabinofuranosyl pyridinium salts are substrates, and an approximate beta lg(V) value of -0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 X 10(9), is comparable with that observed with pyranosidases. 10. Ring-opening mechanisms can therefore be dismissed, even though they are known in the acid-catalysed hydrolysis of arabinofuranosides.

Arabinose↗

PubMed1987

Inactivation of GM1-ganglioside beta-galactosidase by a specific inhibitor: a model for ganglioside storage disease.

This study was designed to establish an in vitro model with biochemical and morphological similarities to the human neurodegenerative disease GM1 gangliosidosis. Utilizing a specific inactivator of the lysosomal enzyme GM1-ganglioside beta-galactosidase (beta-D-galactopyranosylmethyl-p-nitrophenyltriazene [beta-GalMNT]) and neuroblastoma X glioma hybrid cells (NG108-15), we suppressed beta-galactosidase activity for up to 72 hours. Coincidental with suppression of this enzyme to levels less than 1% of control, we found up to a nine-fold accumulation of its substrate, the GM1-ganglioside, and the ultrastructural appearance of membranous cytoplasmic bodies. beta-GalMNT treatment suppressed growth but had little effect on the specific activity of choline acetyltransferase, lactate dehydrogenase, or other lysosomal enzymes including galactosylceramidase. This model should permit studies of the neurophysiological effects of increased ganglioside accumulation and their reversibility.

Brain↗

PubMed1985

Effects of deuterium substitution alpha and beta to the reaction centre, 18O substitution in the leaving group, and aglycone acidity on hydrolyses of aryl glucosides and glucosyl pyridinium ions by yeast alpha-glucosidase. A probable failure of the antiperiplanar-lone-pair hypothesis in glycosidase catalysis.

Neither kcat. nor kcat./Km for five aryl alpha-D-glucopyranosides correlates with aglycone pKa, and isotope effects, described according to the convention used by Cleland [(1982) CRC Crit. Rev. Biochem. 13, 385-428], of 18(V) = 1.002 +/- 0.008, alpha D(V) = 1.01 +/- 0.04 and alpha D(V/K) = 0.969 +/- 0.035 are observed for p-nitrophenyl, and one of beta D(V) = 1.02 +/- 0.04 for phenyl alpha-D-glucopyranoside; kcat. but not kcat./Km, correlates with aglycone pKa for five alpha-D-glucopyranosyl pyridinium ions with a Brønsted coefficient of -0.61 +/- 0.06, and isotope effects of alpha D(V) = 1.22 +/- 0.02, beta D(V) = 1.13 +/- 0.01 and alpha D(V/K) = 1.018 +/- 0.046 for the 4-bromoisoquinolinium, and alpha D(V) = 1.15 +/- 0.02 and beta D(V) = 1.085 +/- 0.011 for the pyridinium salts are observed. These data require that a non-covalent event, fast in the case of the N-glycosides but slow in the case of the O-glycosides, precedes bond-breaking, and that bond-breaking involves substantial charge development on the glycone and near-perpendicularity of the C2-H bond to the planar oxocarbonium ion system. A model meeting these requirements is that the non-covalent event is a conjoint change of protein and substrate conformation which puts the pyranose ring in the 2,5B conformation of the bond-breaking transition state. This model also explains the contrast between the powerful inhibition of the enzyme by deoxynojirimycin (Ki = 23 +/- 3 microM) and feeble inhibition by castanospermine [Saul, Chambers, Molyneux & Elbein (1983) Arch. Biochem. Biophys. 221, 593-597], but is directly contrary to the predictions of Deslongchamps' 'Theory of Stereoelectronic Control' [Deslongchamps (1975) Tetrahedron 31, 2463-2490; (1983) Stereoelectronic Effects in Organic Chemistry, p. 39, Pergamon Press, Oxford].

Binding Sites↗

PubMed1985

Goat liver beta-mannosidases: molecular properties, inhibition and inactivation of the lysosomal and nonlysosomal forms.

The two caprine hepatic beta-mannosidases have been partially purified and their properties have been compared. The lysosomal beta-mannosidase A had an apparent molecular weight of 127,000 +/- 10,000 and an isoelectric point of pH 6-7. Its activity was unaffected by incubation with Triton X-100 (0.1%) and cysteine (20 mM) and it hydrolyzed the presumed natural substrates, Man(beta 1-4)GlcNAc and Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc. The nonlysosomal beta-mannosidase B had an apparent molecular weight of 43,000 +/- 2,000 and an isoelectric point of pH 5.5. beta-Mannosidase B was activated by Triton X-100 (0.1%) and was inhibited by cysteine (20 mM). Hydrolysis of Man(beta 1-4)GlcNAc, but not of Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, followed incubation with beta-mannosidase B. 1,5-Dideoxy-1,5-imino-D-mannitol did not inhibit the A enzyme and only feebly (Ki = 0.3 mM) inhibited the B enzyme; beta-D-mannopyranosylmethyl p-nitrophenyl triazene did not inactivate either enzyme but 1,2-anhydro-1,2,3,5,6/4-cyclohexane hexol inactivated the B enzyme only. The radical mechanistic differences between the two enzymes argue against their having the same genetic origin.

Animals↗

PubMed1984

On the antiperiplanar lone pair hypothesis and its application to catalysis by glycosidases.

The idea that sp3 lone pairs of electrons on a heteroatom direct the departure of a leaving group from an adjacent tetrahedral carbon centre is shown to be untenable as a general principle, and the permissive evidence in favour of it to be accounted for by the principle of least nuclear motion. The application of the idea to the majority of glycopyranosidases, which work by a double-displacement mechanism, is shown to require implausible contortions of the pyranose ring.

Chemical Phenomena↗

PubMed1983

Competitive inhibition of the inverting beta-xylosidase of Bacillus pumilus 12 by monosaccharide derivatives of different structural and conformational types. A possible natural substrate.

The title enzyme is competitively inhibited by compounds, for example alpha-D-xylopyranosylpyridinium salts and 1,6-anhydro-D-glucopyranose, for which the normal 4C1 conformation of the xylopyranose ring is precluded. It is competitively inhibited by compounds, for example beta-D-xylopyranosylpyridinium salts and 1,6-anhydro-L-idopyranose, for which the 1C4 conformation is precluded, and which have no accessible conformations in common with the first set of inhibitors. It is also competitively inhibited by alpha-L-arabinofuranosides. Inhibition by 1,6-anhydroglucopyranose, 1,6-anhydro-L-idopyranose and L-arabinono-gamma-lactone is competitive with respect to each other. alpha-D-Xylopyranosyl fluoride is not a detectable substrate, by itself or in the presence of a representative of any of the three types of inhibitor. On the basis of these and literature data, it is proposed that the natural substrate is a hemicellulose fragment containing the D-Xylp beta (1 leads to 4)-[L-Araf alpha (1 leads to 3)]D-Xylp structure. Tentative inferences about the catalytic mechanism can also be drawn.

Bacillus↗

PubMed1982

Affinity labeling via deamination reactions.

An electrophilic center at saturated carbon generated by the departure of molecular nitrogen shows minimum discrimination between various nucleophiles. The generation of such a center in the active site of a protein is therefore an attractive way of labeling that active site. The chemistry of deamination reactions will be discussed with respect to the practicality of triggering the deamination in the active sites of proteins. Successful applications of this principle using the N-nitrosamide functionality, the alkyl aryl triazene functionality, and the diazo functionality will be described. Reasons why active-site reagents incorporating this type of covert electrophilicity are more specific than those incorporating an overtly electrophilic center (such as -CO-CH2-Halogen) will be advanced. The actual and potential application of deamination precursors to the specific inhibition of physiological activities in living cells will be discussed.

Affinity Labels↗

PubMed1981

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).

Binding Sites↗

PubMed1980

Specific inactivation of lysosomal glycosidases in living fibroblasts by the corresponding glycosylmethyl-p-nitrophenyltriazenes.

1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene can specificag detectable changes in cell viability or the activities of other hydrolases. 2. beta-D-Glucopyranosylmethyl-p-nitrophenyltriazene behaves similarly towards lysosomal beta-glucosidase, but also inactivates some alpha-glucosidase. 3. Both beta-galactosidase and beta-glucosidase activities in triazene-treated confluent fibroblast cultures recover exponentially; if zero-order enzyme production is assumed, turnover times of 10 and 5 days respectively can be estimated.

Cells, Cultured↗