Search PubMed⌕ Search

Biomedical subjects

P M Horowitz

Publications and source records attributed to P M Horowitz.

At least 73 records · Page 4Linked to original sources
PubMed1994

The sulfurtransferase activity and structure of rhodanese are affected by site-directed replacement of Arg-186 or Lys-249.

Two mutants of the enzyme rhodanese that replace Arg-186 with Leu (R186L) or Lys-249 with Ala (K249A) were prepared to test suggestions that these residues are involved in catalysis and structure. The predominant effect with R186L was functional, and Km for the sulfur donor, thiosulfate, increased from 3.7 mM to 73 mM with a modest decrease in Vmax (672 IU/mg to 576 IU/mg). However, K249A was virtually inactive using thiosulfate, but it was active with thiosulfonates such as p-toluene-, 2-aminoethane-, or ethanethiosulfonate, and these compounds could be demonstrated to form persulfide-substituted rhodanese. Compared with wild type enzyme, K249A had (a) reduced stability, (b) comparable secondary structure, (c) more easily exposed hydrophobic surfaces, and (d) a core structure that denatured similarly to the wild type enzyme. Thus, Arg-186 and Lys-249 are important in rhodanese catalysis, and Lys-249 is particularly critical for substrate selectivity and protein stability. Finally, the results suggest that there can be active rhodanese species in vivo that will be undetected using thiosulfate as a sulfur donor.

Amino Acid Sequence↗
PubMed1994

Mutations of noncatalytic sulfhydryl groups influence the stability, folding, and oxidative susceptibility of rhodanese.

Mutants of rhodanese (EC 2.8.1.1) which substitute serine residues for each of the 4 cysteine residues have been studied to determine the roles of cysteines in the structure and function of the enzyme. The proteins compared in these studies were: the wild-type, C63S, C247S, C254S, C263S, C254S/C263S, and C63S/C254S/C263S. These current studies show that cysteine 247 is the only cysteine required for the activity of the enzyme. Although the other sulfhydryl groups do not participate in sulfur transfer, mutations of the noncatalytic cysteines result in the destabilization of the native structure of the enzyme. All the active proteins had similar kinetic parameters. Mutants substituting cysteine 254, compared with the other species, were: (a) more resistant than wild-type to inactivation by dithiothreitol, (b) more readily reactivated following oxidative inactivation, and (c) found to adopt conformations that show increased exposure of hydrophobic surfaces following removal of the transferable sulfur. On the other hand, cysteine to serine substitutions had very little effect on: (a) the rates of oxidative inactivation, (b) the increased fluorescence following the removal of transferable sulfur, or (c) the effectiveness of spontaneous refolding after urea denaturation. Forms of rhodanese that were formerly considered to be irreversibly oxidized can be reactivated if the protein is denatured in urea before reductants are used. It is proposed that these forms differ from reversibly oxidized states due to the inaccessibility of intramolecular disulfides to reductants and not to the formation of higher oxidation states of the protein.

Amino Acid Sequence↗
PubMed1994

Alteration of the quaternary structure of cpn60 modulates chaperonin-assisted folding. Implications for the mechanism of chaperonin action.

Chaperonin-mediated, in vitro folding of rhodanese by the intact protein cpn60 has previously been shown to require cpn10 and ATP hydrolysis (Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991) Nature 352, 36-42; Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M. (1991) J. Biol. Chem. 266, 13044-13049). The present work demonstrates that the rhodanese-cpn60 complex can be dissociated by urea to allow folding to proceed, thus removing the obligatory requirement for cpn10 and ATP. Analytical ultracentrifugation and circular dichroism show that tetradecameric cpn60 can be disassembled into monomers that retain substantial secondary structure. Unfolded rhodanese induces the reassembly of tetradecameric cpn60 from monomers, and binding of rhodanese stabilizes cpn60 quaternary structure. Intermediate cpn60 species, possibly heptamers, are detected at intermediate urea concentrations after addition of unfolded rhodanese. The use of urea has demonstrated a functionally related loosening of subunit interactions in cpn60 that is not detectable under usual solution conditions. Our data suggest a highly dynamic role for the quaternary structure of cpn60 in chaperonin-mediated protein folding.

Adenosine Triphosphatases↗
PubMed1994

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.

Anilino Naphthalenesulfonates↗
PubMed1994

The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide.

The in vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by the E. coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1-23) of rhodanese. In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction. In contrast, this process is substantially inhibited in the presence of the peptide. The maximum recovery of active enzyme is peptide concentration-dependent. The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex. In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery. Further, the unassisted refolding of rhodanese is also inhibited by the peptide. Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme. A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11-23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme. The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites.

Amino Acid Sequence↗
PubMed1993

Physical characterization of a reactivatable liposome-bound rhodanese folding intermediate.

Recently, we described the formation of a complex between liposomes and the unfolded protein rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), which could be liberated and efficiently reactivated after treatment of the complex with detergents [Zardeneta, G., & Horowitz, P. M. (1992) Eur. J. Biochem. 210, 831-837]. Previous data suggested that liposome-bound rhodanese was in the form of a folding intermediate. We have characterized in greater detail the nature of the conformation of the bound rhodanese. Physical characterization of the bound rhodanese intermediate was carried out using proteolysis, fluorescence studies with 1,8-anilinonapthalene-8-sulfonic acid, a probe for hydrophobic site exposure, intrinsic fluorescence to determine tryptophan accessibility using the quenchers acrylamide and iodide, and circular dichroism to detect extent of secondary structure. These studies show that the rhodanese intermediates bound to either cardiolipin or phosphatidylserine liposomes are not identical, the former being in a less compact conformation yet having more secondary structure than the latter, an observation which may explain why the reactivation of the former intermediate is more effective. Finally, turbidimetric and proteolytic studies raise the possibility that each rhodanese intermediate binds to several liposomes. This finding suggests that a possible reason for the differential reactivation yields obtained may be due to the fact that unfolded rhodanese has more binding sites for cardiolipin than for phosphatidylserine liposomes. A greater number of binding sites would result in better anchoring of rhodanese's interactive surfaces and thus reduce the likelihood of misfolding.

Anilino Naphthalenesulfonates↗
PubMed1993

Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding.

When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.

Animals↗
PubMed1993

The folding and stability of rhodanese are influenced by the replacement of glutamic acid 17 in the NH2-terminal helix by proline but not by glutamine.

Two site-directed mutants of the enzyme rhodanese which replace glutamic acid 17 with either glutamine (E17Q) or with proline (E17P) were produced and purified. Both mutants displayed specific activities similar to the wild type enzyme. E17Q was equivalent to the wild type enzyme in all assayed characteristics, except that the mutant had slightly more solvent exposure of hydrophobic surfaces. Results with E17Q suggest that the charge on Glu17 is not required for helix stabilization, nor is its titration required for the low pH structural transitions seen previously. In contrast, E17P was significantly different from the wild type enzyme. For example, E17P had (a) higher exposure of hydrophobic surfaces in the unperturbed state; (b) considerably lower stability to perturbation by urea; (c) easier exposure of organized hydrophobic surfaces on initial unfolding, even though denaturation to the final disorganized state was the same as for the wild type; (d) the ability to refold without assistants but with lower yields and somewhat slower folding; and (e) similar susceptibility to trypsin and evidence of a new clip site closer to the NH2 terminus. However, E17P and the wild type enzyme had very similar recoveries with chaperonin-assisted refolding, and the chaperonin protein groEL had a very similar ability to suppress unassisted refolding. These results indicate that changes in the NH2-terminal sequence can have dramatic effects on the stability of rhodanese and on its ability to be refolded in the absence of assistants. They further suggest that interactions with chaperonins do not rely exclusively on the detailed conformation at the NH2 terminus. A model that incorporates observations here includes step(s) in which the NH2-terminal sequence folds onto the NH2-terminal domain late in the folding process after the protein had adopted a near native conformation.

Amino Acid Sequence↗
PubMed1993

GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation.

Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli. The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed. Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides. The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Bacterial Proteins↗
PubMed1993

Interactive intermediates are formed during the urea unfolding of rhodanese.

Structural transitions have been studied on the pathway for urea denaturation of rhodanese. Unlike guanidinium hydrochloride, urea gives no visible precipitation. Increasing urea concentrations cause a transition in which the enzyme activity is completely lost by 4.5 M urea, and there is a shift of the intrinsic fluorescence maximum from 335 nm for the native enzyme to 350 nm. There is a maximum exposure of organized hydrophobic surfaces at 4.5 M urea as reported by the fluorescence of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid. Above 4.5 M urea, this probe reports the progressive loss of organized hydrophobic surfaces. The polarization of the intrinsic fluorescence falls with increasing urea concentrations in a complex transition showing that rhodanese flexibility increases in at least two phases. Rhodanese becomes increasingly susceptible to digestion by subtilisin between 3.5 and 4.5 M urea, giving rise to large fragments. At urea concentrations > 5 M, rhodanese is completely digested. There is a small increase in the rate of sulfhydryl accessibility between 3.5 and 4.5 M urea, but there is a large increase in the sulfhydryl accessibility above 4.5 M urea. Dimethyl suberimidate cross-linking shows the presence of associated species in 3-5 M urea, but there are few cross-linkable species at lower or higher urea concentrations. These results are consistent with a model in which urea unfolding of rhodanese is associated with the initial production of a species having organized regions of structure with exposed hydrophobic surfaces separated by flexible elements.

Animals↗
PubMed1993

Partially folded rhodanese or its N-terminal sequence can disrupt phospholipid vesicles.

Rhodanese (thiosulfate cyanide sulfurtransferase; E.C. 2.8.1.1) is a mitochondrial enzyme that is unprocessed after import. We describe in vitro experiments showing that partially folded rhodanese can interact with lipid bilayers. The interaction was monitored by measuring the ability of rhodanese to disrupt small unilamellar vesicles composed of phosphatidylserine and to release 6-carboxyfluorescein that was trapped in the liposomes. Partially folded rhodanese, derived by dilution of urea-unfolded enzyme, efficiently induced liposome leakage. Native rhodanese had no effect on liposome integrity. Liposome disruption progressively decreased as rhodanese was given the opportunity to refold or aggregate before introduction of the liposomes. A synthetic 23 amino acid peptide representing the N-terminal sequence of rhodanese was very efficient at disrupting the liposomes. Shorter peptides chosen from within this sequence (residues 11-23 or residues 1-17) had no effect on liposome disruption. A peptide representing the tether region that connects the domains of the enzyme was also without effect. These results are consistent with the hypothesis that the N-terminal sequence of rhodanese is an uncleaved leader sequence, and can interact with membrane components that are involved in the mitochondrial uptake of this protein.

Amino Acid Sequence↗
PubMed1993

Thermally perturbed rhodanese can be protected from inactivation by self-association.

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30-40 degrees C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 micrograms/ml is inactivated at 40 degrees C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 micrograms/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30-40 degrees C. Light scattering kinetics at 40 degrees C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions; at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Glutaraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40 degrees C but not at 23 degrees C. Precipitates formed at 40 degrees C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.

Disulfides↗
PubMed1992

Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate.

The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV-sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, beta-mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interact directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation-competent prefolded conformation, which may readily lead to a correctly folded enzyme.

Animals↗
PubMed1992

Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese.

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.

Acrylamide↗
PubMed1992

Analysis of the perturbation of phospholipid model membranes by rhodanese and its presequence.

The ability of the cytoplasmically synthesized mitochondrial enzyme rhodanese and its putative import signal sequence to interact with model phospholipid membranes was characterized. Membrane perturbation assays were used to test a current hypothesis that the initial step in protein translocation may involve binding of signal sequences with membrane lipids. Here we show comparative studies on the effect of native and various forms of denatured rhodanese, as well as two peptides, rho(1-23) and rho(11-23), derived from its NH2-terminal sequence, on the perturbation of 6-carboxyfluorescein-containing large unilamellar vesicles composed of either cardiolipin, phosphatidylcholine, or phosphatidylserine. We monitored the degree of perturbation by measuring dye leakage and found differential perturbation by either peptide or protein. Unfolded rhodanese perturbed vesicles in the order phosphatidylserine > cardiolipin >> phosphatidylcholine. Denatured rhodanese was approximately 25 times more effective (on a molar basis) than rho(1-23) in the disruption of anionic liposomes. Rho(11-23) was unable to perturb liposomes. We found an inverse correlation between degree of activity of rhodanese folding intermediates and their ability to perturb liposomes. On urea denaturation, enzymatic activity was completely lost before membrane perturbation ability reached significant levels. Analysis of the peptides by circular dichroism showed that anionic liposomes can induce alpha-helical structure only in rho(1-23) and denatured rhodanese. Intrinsic peptide fluorescence studies showed that only rho(1-23) and denatured rhodanese partitioned into these model membranes. Results obtained here imply that peptides from naturally occurring alpha-helical structures may need adjacent motifs for helical structure induction in lipid environments, and the subsequent secondary structure may, in turn, promote partitioning of these segments into the lipid phase and ultimately lead to membrane perturbation.

Amino Acid Sequence↗
PubMed1992

Reducing sugars can induce the oxidative inactivation of rhodanese.

The enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is inactivated on incubation with reducing sugars such as glucose, mannose, or fructose, but is stable with non-reducing sugars or related polyhydroxy compounds. The enzyme is inactivated with (ES) or without (E) the transferable sulfur atom, although E is considerably more sensitive, and inactivation is accentuated by cyanide. Inactivation of E is accompanied by increased proteolytic susceptibility, a decreased sulfhydryl titer, a red-shift and quenching of the protein fluorescence, and the appearance of hydrophobic surfaces. Superoxide dismutase and/or catalase protect rhodanese. Inactive enzyme can be partially reactivated during assay and almost completely reactivated by incubation with thiosulfate, lauryl maltoside, and 2-mercaptoethanol. These results are similar to those observed when rhodanese is inactivated by hydrogen peroxide. These observations, as well as the cyanide-dependent, oxidative inactivation by phenylglyoxal, are explained by invoking the formation of reactive oxygen species such as superoxide or hydrogen peroxide from autooxidation of alpha-hydroxy carbonyl compounds, which can be facilitated by cyanide.

Fructose↗
PubMed1992

Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.

Animals↗