The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase

The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.     

Altered responsiveness to parasympathetic activation of submaxillary salivary gland in the heat-acclimated rat

Submaxillary salivary gland responsiveness during the heat acclimation procedure (34 +/- 1 degree C) was studied in the rat. Gland responsiveness was evaluated by measuring saliva flow rate of the anesthetized animals following either parasympathetic nerve stimulation or i.v. application of pilocarpine. A thirty percent decrease in glandular responsiveness for the two modes of stimulation was measured during the first 10 days of acclimation. Following 60 days of acclimation recovery was observed. It is concluded that decreased responsiveness of the heat-acclimated gland is a glandular event. Increased saliva flow occurring at the initial phase of acclimation is apparently due to changes in the thermoregulatory center.     

Redistribution of cardiac output in anesthetized thermally dehydrated rats

Cardiac output (CO) and its distribution were studied in dehydrated (37 degrees C) anesthetized (Na thiopentone) rats prior to and following heat acclimation (at 34 degrees C), using 57Co 15 micron microspheres. In non-acclimated dehydrated rats, CO decreased while heart rate (HR) increased significantly. Following acclimation CO increased without any change in HR; during dehydration CO remained elevated together with a significant increase in HR. In non-acclimated rats at low dehydration blood perfusion to peripheral thermoregulatory areas increased while perfusion of splanchnic area decreased; at high dehydration level peripheral blood flow decreased whereas splanchnic blood flow was augmented. In acclimated dehydrated rats, CO distribution to thermoregulatory areas did not change while perfusion of the splanchnic area decreased. It is suggested that following acclimation, the increased CO contributes to maintenance of thermoregulatory peripheral blood flow; in non-acclimated rats severe dehydration leads to augmented blood flow in the permeable splanchnic vascular bed, increasing efflux of plasma protein and failure of plasma volume conservation.     

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.     

Nucleotide and Mg2+ Induced Conformational Changes in GroEL can be Detected by Sulfhydryl Labeling

The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the molecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg²⁺ and/or adenine nucleotides. Three peptides, each containing one of the cysteines of GroEL (C138, C458 and C519) were identified. GroEL labeled in 50mM TrisHCl, pH 7.8, incorporated ~0.3 labels each on C138 and C458. With 10mM MgCl₂, the labeling increased to ~0.8 labels each on C138 and C458. The increase was partially due to a conformational change which occurred upon Mg²⁺ binding as well as to an increase in ionic strength. When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg²⁺, C138 incorporated ~0.8 labels, while C458 incorporated ~0.1 labels. These results suggest that the binding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible. GroEL slowly dissociated into monomers when it was extensively labeled at C458. GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was able to bind but not release active rhodanese. The observed variations in sulfhydryl accessibility are consistent with mechanisms that suggest binding of GroES to GroEL differs from the binding of substrate protein to GroEL, and that the binding of Mg²⁺ or Mg-adenine nucleotides results in conformational changes in GroEL.