Titration Of SH Groups Research Articles

Anti-aggregation properties of trehalose on heat-induced secondary structure and conformation changes of bovine serum albumin

During our experimental work, aggregation of bovine serum albumin was obtained incubating the protein solution at 60 °C to investigate temperature-induced secondary structure, conformation changes and anti-aggregative activity of trehalose. IR-measurements suggested that in the presence of 1.0 M of trehalose there is a little increase in short segment connecting α-helical and a clearly decrease in the loss of α-helix structure and in the formation of intermolecular and antiparellel β-sheet up to 78 and 55%, respectively. Useful information also arose following the temperature evolution of Amide I′ band profile in the range of temperature between 25 and 90 °C in absence or in presence of 1.0 M trehalose. Complementary information is obtained by electrophoresis, circular dichroism, fluorescence spectroscopy, titration of SH groups and light scattering measurements. Results encouraged biotechnology and pharmaceutical application of the disaccharide and provided evidence for its utilization in degenerative diseases evolving via aggregation process.

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0–3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 °C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV–vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.

Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins.

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.

Chemical Modification of SH Groups of E. coli Phosphofructokinase-2 Induces Subunit Dissociation: Monomers Are Inactive but Preserve Ligand Binding Properties

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive. However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP. The stoichiometry of the label incorporation was found to be 2.03 ± 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5′-dithiobis(2-nitrobenzoic acid) in the labeled protein. HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer–tetramer transition is promoted. In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation. Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme. Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern–Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP–enzyme complexes. These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity.

Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

Inhibition of [formula omitted]-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation

In the present work, we studied the effects of phenoxyl radicals, generated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na + K + -ATPase by characterizing interactions of VP-16 phenoxyl radicals with the enzyme's SH-groups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na + K + -ATPase which was dependent on the incubation time and concentration of tyrosinase. The inhibition of Na + K + -ATPase was accompanied by a decrease of DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)-titratable SH-groups. In the presence of Na + K + -ATPase, a typical ESR signal of the VP-16 phenoxyl radical could be observed only following a lag period the duration of which was proportional to the concentration of the Na + K + -ATPase added. Our HPLC measurements demonstrated that Na + K + -ATPase protected VP-16 against tyrosinase-catalyzed oxidation. Combined these results suggest that redox-cycling of VP-16/VP-16 phenoxyl radical by SH-groups of Na + K + -ATPase occurred. Ascorbate which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-groups. Reduction of VP-16 phenoxyl radicals by ascorbate was directly observed by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na + K + -ATPase may be responsible for at least some of its clinical side effects (e.g., cardiotoxicity) which can be prevented by ascorbate.

Determination and Derivatization of Protein Thiols by n-Octyldithionitrobenzoic Acid

n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman′s reagent, can be isolated in crystalline form. Like Ellman′s reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman′s reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman′s reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.

Temperature-induced conformational changes in turtle myoglobin

A sea turtle myoglobin has been spin labelled in the cyanomet form (MbT-CN) with a maleimide spin label and temperature-induced changes were monitored by EPR. The EPR spectrum is composed by an immobilized and a more mobile signal. Comparison of this spectrum with spectra obtained from other proteins, blocking of the NH2-terminal and titration of SH groups lead to the conclusion that a reactive histidine is involved in the spin labelling. Our results on the temperature-induced changes monitored by the maleimide spin label suggest that a sequence of events is taking place in the transition which leads to protein denaturation (predenaturation): with increase in temperature atpH 8.5 the EPR parameter for the immobilized signal (α EPR) is the first to report the structural change (T 1/2=28°C); then the mobile signal changes (T 1/2=44°C) and finally the active centre monitored by optical absorption of the iron heme undergoes these changes (T 1/2=64°C). The activation energy calculated from the mobile spectrum is around 3 kcal/mol showing that these changes are quite subtle if compared to the enthalpy changes monitored by optical absorption of iron (40 kcal/mol). This predenaturation occurring in the range (30÷50)°C seems to be a general property of myoglobins, as has been observed also for whale and applysia.

Inhibition by lysine and arginine of the conversion of C3 and B in the serum and a purified system

When human serum was incubated at 45°C for 30 min, C3 and B were converted to C3b and Bb. Molecular weights of purified C3 and B were shown not to change after incubation at 50°C. Spectropolarimetry indicated that the secondary structures of C3 and B changed after incubation at higher temperature. The titration of SH groups in the C3 molecule showed the liberation of an SH group. These results show that the alternative complement pathway is activated at raised temperatures without known activators such as zymosan or lipopolysaccharide (LPS). This may be due to the accelerated interaction of conformationally changed components of the alternative pathway such as C3 and B. Using this system, effects of various substances on the interaction of C3 and B in serum and the purified system were investigated. The addition of arginine and lysine resulted in the inhibition of the conversion of C3 and B in the serum at elevated temperature. Other amino acids such as anionic amino-acids and NaCl did not influence the conversion. In the purified system, only arginine and lysine prevented the conversion of C3 and B, when C3, B and D were incubated in the presence of Mg ++ and amino-acids. Since lysine and arginine did not inhibit the enzymatic activity of D , these data suggest that arginine and lysine prevent the interaction of C3 and B in the serum at elevated temperatures.

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl −-dependent, ouabain-resistant K + transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl −-dependent K + fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb + as a K + analogue. In contrast to intact cells, the rate constants of the base level Rb + efflux in ghosts were similar in NaNO 3 and NaCl (° k Rb = 0.535 ± 0.079 h −1 and 0.534 ± 0.085 h −1, respectively), while 1 mM N-ethylmaleimide stimulated Rb + efflux strongly in NaNO 3 (° k Rb = 14.26 ± 1.32 h −1) and moderately in NaCl (° k Rb = 2.73 ± 0.54 h −1). This effect was dependent on the presence of internal ATP. Stimulation of Rb + efflux was observed in the presence of ⩾ 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb + efflux was approx. 50% inhibited by 100 μM quinine sulfate whereas 1 μM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb + concentration, but rates increased linearly in NaNO 3. Replacement of external Na + with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb + efflux, suggesting a role for external Na +. N-Ethylmaleimide-stimulated Rb + efflux was greater in buffers with lipophilic anions such as SCN − or NO 3 − than in solutions with Cl − or acetate. However, the conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb + influxes in intact red cells.

Active site-directed modification of tryptophanase by 3-bromopyruvate.

Tryptophanase purified from Escherichia coli B/It7-A was irreversibly inactivated by 3-bromopyruvate following pseudo-first-order kinetics. The inactivation rate for the holoenzyme tended to saturate as the concentration f bromopyruvate increased. L-Alanine and DL-3-phenylserine, potent competitive inhibitors with respect to L-tryptophan decomposition, protected the enzyme from inactivation. Titration of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of one SH group per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to bromopyruvate-modification following pretreatment with DTNB, the activity was almost completely restored upon reduction with dithiothreitol. Modification of the enzyme with bromopyruvate quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results support the possibility that bromopyruvate reacts with the enzyme as an affinity labeling agent.

PH-induced unfolding of the constant fragment of the immunoglobulin light chain: effect of reduction of the intrachain disulfide bond.

Circular dichroism and proton nuclear magnetic resonance measurements were made to clarify how reduction and reduction plus alkylation of the intrachain disulfide bond affect the conformation and stability of the constant (CL) fragment of the immunoglobulin light chain. The pH titration behavior of the two histidine residues in the intact CL, reduced CL, and reduced and alkylated CL fragments were followed by proton nuclear magnetic resonance spectroscopy. It was shown that reduction of the intrachain disulfide bond does not affect the solution conformation of the immunoglobulin fold, whereas reduction and alkylation results in extensive unfolding of the protein molecule. These results are consistent with the previous results obtained by Goto and Hamaguchi [1979) J. Biochem. 86, 1433-1441) using circular dichroism, fluorescence, and titration of SH groups. The pH-induced unfolding of the reduced CL fragment was compared with that of the intact CL fragment. The stability of the intact CL fragment to acid was much greater than that of the reduced CL fragment. The equilibria of the unfolding by acid could be explained by assuming that the same ionizable groups participate in the unfolding for the reduced CL fragment as for the intact CL fragment, and that the stability of the intact CL fragment is 100 times greater than that of the reduced CL fragment. In the alkaline pH region, the reduced CL fragment was unfolded above pH 8.5, while the intact CL fragment was not unfolded until pH 11.6. The kinetics of the unfolding by alkali of the reduced CL fragment was also studied. The unfolding by alkali of the reduced CL fragment could be explained by assuming that the reduced CL molecule can no longer adopt the folded conformation when either or both SH groups ionize.

The inactivation of thymidylate synthase by periodate

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of 10 4 − at 0°C Nearly complete inhibition resulted when the IO 4 − concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO 4 − was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by 10 4 −, with the order of effectiveness dUMP > dTMP > phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by 10 4 −. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercunbenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with penodate could be completely reactivated with ME or DTT.

Binding of Chlorpromazine to S‐100 Protein

Chlorpromazine (CPZ) induces in S-100 conformational changes resulting in the exposure of titratable SH groups of the protein to the solvent. This effect is even greater in the presence of Mg2+ +/- Ca2+. S-100 possesses binding sites for CPZ. The binding of CPZ to 3 microM S-100 is half-saturated by 0.18 microM CPZ in the presence of Mg2+ plus Ca2+ and by 0.24 microM CPZ in the presence of Mg2+ plus EGTA. The extent of the binding is greater in the presence of Ca2+ than in the presence of EGTA, especially at low CPZ concentrations.

Effect of Metal Ions on the Structure and Activity of Calcium-Activated Neutral Protease (CANP)1

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.

Activity and properties of ribulose‐1,5‐bisphosphate carboxylase of sugarbeet plants grown under saline conditions

Activity and properties of sugar beet (Beta vulgaris var. Polyrave) leaf ribulose‐1,5‐bisphosphate (RuBP) carboxylase were investigated following the exposure of plants to NaCl in the range of 45 to 270 mM for 7 days. An enhancement in RuBP carboxylase activity was found both in crude extracts and in purified preparations following plant exposure to 180 mM NaCl. Kinetic properties of the enzyme were significantly affected by salinity as determined by a 4.5 fold increase in Km [HCO‐3] and Km [CO2], and a Vmax increase of 50%. Data based on polyacrylamide‐gel‐electrophoresis suggest that the molecular weight of the small subunit of RuBP carboxylase was reduced from 15,500 to 12,500 in plants grown under salinity. The large subunit was much less affected and no change was found in the whole enzyme. The enzyme isolated from plants exposed to salinity contained about 50% fewer titratable SH groups as compared with the control. The results indicate that in this plant, mild salt concentrations induced conformational changes in RuBP carboxylase which may be responsible for its tolerance to semi‐salinity.

Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo.

The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).

Characterization of liver prenyl transferase and its inactivation by phenylglyoxal

The two interconvertible forms of pig liver prenyl transferase, A and B, consist of two identical subunits of M r = 38 500 and are dimers. Form A contains six titratable SH-groups, whereas form B contains only four per dimer. The amino acid composition of the two forms is otherwise identical. Both enzyme forms are inactivated by phenylglyoxal. The inactivation in the absence of Mg 2+ or Mn 2+ is biphasic, each phase following pseudo first-order kinetics and is accompanied by a proportional binding of [ 14C]phenylglyoxal to the protein. In the initial fast phase of inactivation ( t 1 2 = 9.6 min ) the amount of [ 14C] phenylglyoxal bound to the enzyme extrapolated to 1.1 arginyl residues and in the second phase ( t 1 2 = 23 min ) to 2.2 arginyl residues modified per subunit for complete inactivation. 1 mM Mg 2+ and 0.1 mM Mn 2+ abolished the initial fast rate of inactivation and reduced its rate to a single half-life of about 60 min. Even at this slow rate of inactivation in the presence of Mg 2+, the amount of [ 14C] phenylglyoxal bound to the enzyme extrapolated to about 2.3 arginyl residues modified per subunit for complete inactivation. In the absence of Mg 2+ or Mn 2+ only 1 mM geranyl pyrophosphate protected the enzyme against inactivation. However, in the presence of 1 mM Mg 2+, isopentenyl, dimethylallyl and geranyl pyrophosphates gave additional protection over that observed with the metal ions, geranyl pyrophosphate being the most effective at 0.1 mM concentration.