Jose A. Mendoza

Recombinant bovine rhodanese : purification and comparison with bovine liver rhodanese

Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

Tetradecameric chaperonin 60 can be assembled in vitro from monomers in a process that is ATP independent

The present work shows that monomers of cpn60 (groEL) formed at 2.5 M urea could be assembled to tetradecamers in a process that was independent of ATP. Reassembled cpn60 was able to assist the folding of urea unfolded rhodanese. When cpn60 was incubated at urea concentrations higher than 2.75 M, assembly of tetradecameric cpn60 did not occur after dialysis, and the presence of ATP did not stimulate the assembly process. The cpn60 used here did not display the previously reported ATP-dependent self-assembly of cpn60 monomers that required a higher urea concentration (4 M) for formation (Lissen et al. (1990) Nature 348, 339-342). Assembly and disassembly of cpn60 tetradecamers were followed as a function of the urea concentration by ultracentrifugation and gel electrophoresis in the presence of urea. The electrophoresis results demonstrate that there is rapid assembly of tetradecamers following preincubation and rapid removal of urea at concentrations lower than 2.5 M. Thus, previous methods monitored irreversible dissociation of cpn60, and the present results indicate that the cpn60 assembly requirements for ATP are dependent on pretreatment conditions.

Sulfhydryl modification ofE. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Differential chemical modification ofE. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4′-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.

Differences in herbal and dietary supplement use in the Hispanic and non-Hispanic pediatric populations.

Background: Complementary and alternative medical therapies are becoming increasingly popular in the general population. Objective: To describe the cultural differences in the use of herbal and dietary supplements in the Hispanic and non-Hispanic-Caucasian outpatient pediatric populations. Methods: Questionnaires were administered over a 2-month period to a convenience sample of adolescents and parents of patients younger than 12 years, presenting to an emergency department, an urban private pediatric practice, and a community-based clinic. Results: There were 643 surveys completed. Ethnic distribution was 65% Caucasian, 27% Hispanic, 2% Pacific Islander, and 1% each Asian, African American and Native American. Mean respondent age was 30.8 years. Mean child age was 4.6 years; 51% were male. Use of nonprescribed dietary supplements was significantly greater in Hispanic (33%) versus Caucasian children (9%) (P < 0.01); most commonly used supplements were herbal teas (56%) and echinacea (14%). More Hispanic respondents reported receiving information on herbal preparations from a family member compared with non-Hispanic patients (56.0% vs. 18.7%). Complementary and alternative medicine use had not been discussed with a health care provider by 38% of the total users and 47% of those thought it not important to do so. Conclusions: There is significant use of complementary and alternative medicine in the pediatric population, and herbal and dietary supplement use varies between Hispanic and Caucasian children. In addition, this dietary supplement use is often not discussed with health care providers. These factors should be taken into consideration by all health care providers.

Mutational Analysis of the BPTI Folding Pathway

Over the past three decades, considerable effort has been focused on elucidating the mechanisms by which polypeptide chains fold into well-defined three-dimensional structures (Kim & Baldwin, 1990; Creighton, 1992a; Mattthews, 1993). Major goals of these studies include the identification and characterization of partially folded intermediates and the analysis of transition states that represent the energetic barriers in the folding mechanism. Recently, there has been great progress in the structural analysis of folding intermediates by high resolution NMR spectroscopy of intermediate analogs and native proteins that have been isotopically-labeled during refolding. Structural analysis alone, however, is not sufficient to determine why particular intermediates form or what types of interactions stabilize their conformations. By their very nature, transition states are even more difficult to characterize directly. Questions about folding energetics and the roles of individual interactions in determining the folding mechanism can often be addressed by studying the folding of protein variants that differ by relatively small perturbations of the covalent structure. Recently-developed genetic techniques have made it possible to alter virtually any amino acid residue in a protein, and mutational methods have now been used to study the folding mechanisms of several proteins (Fersht et al., 1992; Goldenberg 1992a; Jennings et al., 1992). We describe here some of our recent work using amino acid replacements to study the folding of a particularly well-characterized protein, bovine pancreatic trypsin inhibitor (BPTI).

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

Thein vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by theE. 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.

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 describein 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.