Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

The behaviour of trypsin towards α-N-methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin

1. alpha-N-Methyl-alpha-N-toluene-p-sulphonyl-l-lysine beta-naphthyl ester (MTLNE) was synthesized as its hydrobromide and shown to be slowly hydrolysed by bovine pancreatic trypsin. The acylation step, however, is so much faster than deacylation of the acyl-enzyme that spectrophotometric measurement of the ;burst' of beta-naphthol provides a convenient method for determining the absolute molarity of trypsin solutions. 2. By using the same stock solution of trypsin, application of this method at pH4.0 and pH7.0 as well as that of Bender et al. (1966) at pH3.7 gave concordant results. 3. Provided that [S](0)>[E](0), the size of the ;burst' is independent of substrate concentration. 4. In the trypsin-catalysed hydrolysis of alpha-N-toluene-p-sulphonyl-l-arginine methyl ester, MTLNE functions as a powerful non-competitive inhibitor. 5. There is no detectable reaction between MTLNE and either bovine pancreatic alpha-chymotrypsin at pH4.0 or bovine thrombin at pH6.0.     

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.     

Binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with Kunitz and Kazal inhibitors as well as the acylating agent p-nitrophenyl p-guanidinobenzoate. A thermodynamic and kinetic study

Thermodynamics and kinetics of binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor), the porcine pancreatic secretory inhibitor (PSTI, Kazal inhibitor) and the acylating agent p-nitrophenyl p-guanidinobenzoate have been investigated at pH 7.4 and 21(+/- 0.5) degrees C. The affinity of both effector dipeptides for bovine trypsinogen: BPTI and bovine trypsinogen: PSTI binary adducts is higher than that observed for the formation of the dipeptide: bovine trypsinogen: p-guanidinobenzoate ternary complexes; moreover, the affinity of Ile-Val for the zymogen binary adducts is higher than that observed for Val-Val association. Binding of Ile-Val and Val-Val to the bovine trypsinogen binary complexes conforms to the induced-fit model, which consists of a fast pre-equilibrium followed by intramolecular isomerization change(s), the latter fast pre-equilibrium followed by intramolecular isomerization change(s), the latter representing the rate-limiting first-order process. For the three bovine trypsinogen systems considered, the rate of the intramolecular isomerization change(s) is essentially independent of the nature of the dipeptide and of the proenzyme binary complex.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of azidobenzamidines as photoaffinity labels for trypsin

Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversible in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-l-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation. Maximum inactivation of trypsin is achieved at pH 6.2 using either azidobenzamidine derivative. Evaluation of the pH dependence of photoaffinity labeling may provide a sensitive tool for probing conformational changes in inhibitor binding sites. These studies provide a basis for the use of azidobenzamidines as photoaffinity analogs of lysine and arginine side chains.     

Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin.

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.     

Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.     

Fluorescence studies on the interaction of dansyl-L-arginine with trypsin and trypsinogen.

The enhancement of fluorescence intensity of the dansyl group due to the formation of trypsin- or trypsinogen-dansyl-L-arginine complex was measured. Dansyl-L-arginine (L-DA) is a product in the trypsin-catalyzed hydrolysis of dansyl-L-arginine methylester. Trypsinogen was found to have only one binding site for L-DA with the dissociation constant of 6.9 x 10(-3)M, which is identical with the Michaelis constant for the trypsin-catalyzed hydrolysis of dansyl-L-arginine amide (Goto, S. and Hess, G.P., unpublished results). This finding and the results of X-ray diffraction studies (1,2) suggest that this binding site is located in the active site of the enzyme. On the other hand, the active enzyme, trypsin, was found to have at least two binding sites for L-DA. One is located in the active site. The dissociation constant for L-DA bound to this site is 6.7 x 10(-3)M. The other site is probably located in the allosteric site of trypsin. The dissociation constant for L-DA bound to this site is 4.8 x 10(-4)M.     

Antithrombic activity of N(alpha)-lauroyl-L-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-arginine methyl ester (Fmoc-L-Arg-OMe)

Simple synthetic compounds of lauroyl-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-agrinine methyl ester (Fmoc-Arg-OMe) were studied for their inhibitory effect on the hydrolysis of chromogenic substrate Tos-Gly-Pro-Arg-pNA (Chromozym TH) by thrombin with K(i) for LAE 1.92 microM and 77 microM for Fmoc Arg-OMe. It was shown that LAE inhibits thrombin activity almost 20 times more strongly than trypsin (K(i) = 18.9 microM). At the same time LAE preserves the ability to be hydrolyzed by thrombin at pH 8.5 (k(cat) = 3.6 c(-1)) and trypsin (k(cat) = 56 c(-1)). It is suggested, that LAE ability to suppress growth of some microorganisms is conditioned to some extent by its ability to inhibit the activity of trypsin-like serine proteases, participating in the infection process.     

Fluorescence and stopped-flow studies on the N in equilibrium F transition of serumalbumin.

In order to characterize the isomerization of serumalbumin in the acidic pH-range equilibrium and kinetic measurements of the intrinsic fluorescence of bovine serumalbumin and human serumalbumin were performed. Additional experiments with modified bovine serumalbumin made use of substituted 1.9 benzoxanthene dyes as SH-specific extrinsic fluorophores. The intrinsic fluorescence (lambda exc = 275 nm) shows a pH-dependent shift of the maximum of fluorescence emissions which correlates with the N in equilibrium F isomerization. This and the acid expansion at pH less than 3.5 is indicated by the pH-dependence of the fluorescence intensity at 350 nm. While tyrosine fluorescence is increased in all steps of the transition, tryptophane fluorescence is decreased in a different way for BSA (2 trp/molecule) and HSA (1 trp/molecule), the latter showing the N in equilibrium F transition only. Combining the tryptophan fluorescence data with the results from the SH-specific modification of BSA the conclusion may be drawn that the tryptophan residues in BSA and the SH-group belong to different domains of the molecule. Stopped-flow experiments prove the N in equilibrium F' and the F' in equilibrium F transitions to be separable along the time axis, the relaxation times being in the range between 40-50 and 300-600 msec respectively. For the "expansion" the kinetic constants critically depend on the initial pH conditions of the solutions. The backward reaction F leads to N seems to be a multistep isomerization process which is characterized by relaxation times greater than 1 sec.     

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.     

Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.     

Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase

Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methotrexate concentration indicates that the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 X 10(7) M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 X 10(5) M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.     

Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labelled proteins

In order to establish optimum conditions for the chemiluminescent (CL) reaction of two acridinium ester labelled proteins (human albumin and rabbit anti-human albumin IgG), we investigated the effects of the following factors known to influence the CL emission: pH, presence of proteins, relative concentrations of components of CL reaction and presence of surfactants. Under optimal conditions of pH and hydrogen peroxide concentration, hexadecyl trimethyl ammonium chloride (CTAC) increased the intensity of the CL reaction of the acridinium ester labelled albumin by 42-fold. Triton X-100, Tween-20, 23 lauryl ether (Brij 35) and sodium dodecyl sulphate (SDS) exerted a much smaller effect. In the case of the acridinium ester labelled antibody, the greatest increase was obtained with Triton X-100 (15-fold) followed by CTAC, Brij 35 and Tween 20 (SDS decreased the emission intensity).     

Low-temperature reactions of trypsin with p-nitroanilide substrates: tetrahedral intermediate formation or enzyme isomerization

The reactions of trypsin with the p-nitroanilides of N alpha-carbobenzoxy-L-lysine, N alpha-carbobenzoxy-L-arginine, and N alpha-benzoyl-L-arginine have been studied in the 0 to -30 degrees C temperature region, over a range of pH values, using 65% (v/v) aqueous dimethyl sulfoxide cryosolvent. At alkaline pH, -30 degrees C, the catalytic reaction appears as a slow "burst" of product (or intermediate) followed by turnover. For all three substrates, the rate of the burst phase is identical. Preincubation of the enzyme at -30 degrees C abolishes the burst. On addition of trypsin to the cryosolvent at -30 degrees C, a time-dependent decrease in fluorescence emission is observed with the same rate as that of the burst with the anilides. The burst phase is thus interpreted as reflecting a temperature/solvent-induced isomerization of trypsin to a less catalytically efficient form, rather than the previously suggested formation of a tetrahedral intermediate [Compton, P. D., & Fink, A. L. (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. The isomerization is not observed at high temperature (greater than or equal to 0 degree C) or at neutral pH. The burst phase was not observed with aqueous methanol cryosolvent, indicating that it is sensitive to both cosolvent and temperature.     

The reaction of bovine thrombin with N-butyrylimidazole. Two different reactions resulting in the inhibition of catalytic activity.

N-Butyrylimidazole has been found to be a potent inhibitor of purified bovine thrombin. The rate and extent of inhibition of thrombin by N-butyrylimidazole could be reduced by the presence of benzamidine, a competitive inhibitor, or by the ester substrate, p-tosyl-L-arginine methyl ester. Spectral studies of the reaction of N-butyrylimidazole with thrombin demonstrated the modification of approximately 1 mol of tyrosine/mol of enzyme at maximum inhibition. In addition to the reaction with tyrosine, N-butyrylimidazole also appears to react with a residue at the "active site" as judged by a decrease in the number of active sites available in the modified enzyme for titration with p-nitrophenyl-p'-guanidinobenzoate. The time course of ester hydrolysis by butyrylated thrombin showed a distinct lag phase suggesting partial reactivation of the enzyme under assay conditions. Partial reactivation of the modified enzyme also occurred spontaneously upon standing in 0.5 M NaCl but was much faster in presence of imidazole (0.03 M, pH 7.6). It is suggested that, in addition to reaction with tyrosine, there is a reaction of N-butyrylimidazole with either the histidine and/or serine residue at the active site of thrombin resulting in a derivative unstable under esterase assay conditions such as that described for the reaciton of N-acetylimidazole with trypsin (L. L. Houston and K. A. Walsh (1970), Biochemistry 9, 156).     

Effects of methanol cryosolvents on the structural and catalytic properties of bovine trypsin

The effect of aqueous methanol cryosolvents on the catalytic and structural properties of bovine trypsin has been investigated. The low freezing points and low viscosities of methanol-based cryosolvents are desirable for a variety of cryoenzymological experiments. Increasing concentrations of methanol caused increases in the values of kcat and Km for the hydrolysis of N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at 0 degrees C and a small increase in Ki for inhibition by benzamidine. Based on product analysis the increase in kcat with increasing methanol concentration at pH* 4.0 and 6.5 can be completely accounted for by nucleophilic competition of methanol for the acyl enzyme intermediate. This observation indicates that deacylation is the rate-limiting step under these conditions. The effect of increasing methanol concentration on kcat/Km for the above ester substrate and N alpha-benzoyl-L-arginine p-nitroanilide was similar. Incubation experiments indicated that trypsin was quite stable in 70% methanol at 0 degrees C and below. The Arrhenius plot for the catalytic reaction in 70% methanol was linear over the 0 to -40 degrees C range, indicating no change in rate-determining step nor temperature-induced structural perturbation. No evidence for structural effects induced by methanol or temperature were detected by monitoring the intrinsic fluorescence and absorbance. We conclude that aqueous methanol cryosolvents are satisfactory for cryosolvent studies of trypsin.     

Isolation, determination of structure and synthesis of the acid-labile conjugate of aldosterone.

1. After administration of 600mg of 3H-labelled aldosterone to human volunteers, 57 mg of homogeneous acid-labile conjugate was isolated from the urine and identified as aldosterone 18 beta-D-glucosiduronic acid. 2. Esterification and acetylation of the conjugate gave a tetra-acetate methyl ester, which, by measurement of the optical rotation and nuclear-magnetic-resonance spectrum, was shown to be a beta-glucosiduronate. This tetra-acetate methyl ester was synthesized in approx. 10% yield by the Koenigs-Knorr procedure. 3. Removal of the acetyl and methyl ester groups from the tetra-acetate methyl ester with alkali was accompanied by almost complete isomerization at C-17 to give 17-isoaldosterone 18 beta-D-glucosiduronic acid. 4. To prevent inversion at C-17 during removal of the acetate and ester groups of beta-glucosiduronate (a) the 3,20-disemicarbazone was prepared, (b) the acetate and ester groups were removed from the disemicarbazone by treatment with alkali, and (c) the semicarbazone groups were removed from the product at pH 2.0, and aldosterone 18 beta-D-glucosiduronic acid was obtained in 47% overall yield. 5. In the presence of components used to synthesize beta-glucosiduronate by the Koenigs-Knorr reaction this substance is converted slowly into the alpha-glucosiduronate; this conversion is responsible, in part, for the low yield of beta-glucosiduronate. 6. Two additional conjugates were obtained in the Koenigs-Knorr reaction; a provisional structure was assigned to one substrate. The other substance is a C-18 alpha-glucosiduronate. Removal of the acetyl and ester groups from C-18 alpha-glucosiduronate gave the alpha-glucosiduronic acid in 84% yield and the 17-isoaldosterone alpha-glucosiduronic acid in 12% yield. 7. The rate at which several types of beta-glucuronidase hydrolyse the foregoing steroidal alpha- and beta-glucosiduronic acids is given.