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A Westcott

Publications and source records attributed to A Westcott.

7 recordsLinked to original sources
PubMed1999

Disulfide bond reduction: A powerful, chemical probe for the study of structure-function relationships in the hemocyanins.

The copper-containing hemocyanins are a class of oxygen-transport proteins whose structures differ in arthropods and molluscs. Crystal structure analyses and amino acid sequence comparisons show that disulfide bonding is a common feature in both arthropod and mollusc hemocyanins. Reduction of the disulfide bonds of a representative set of arthropod and mollusc hemocyanins results in complete loss of their oxygen-binding capacities. Thus, retention of the disulfide bonds is essential to the functional integrity of the oxygen-binding sites in the subunits of this class of oxygen carriers, despite the very different architectures of the arthropod and mollusc molecules. Depending upon the specific hemocyanin, partial to virtually complete restoration of the oxygen-binding capacity occurs when the disulfide-bond reductant is removed by dialysis. The rate at which the functional, active-site geometry is lost and the extent to which it can be restored varies markedly with hemocyanin type, aggregation state, and experimental conditions. Consequently, a comparison of these differences provides a simple, but powerful, way to probe internal and environmental factors that govern physiologically important structure-function relationships in this entire class of oxygen-transport proteins.

Animals↗
PubMed1998

Active-site disruption in native Limulus hemocyanin and its subunits by disulfide-bond reductants: a chemical probe for the study of structure-function relationships in the hemocyanins.

The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins.

Animals↗
PubMed1984

Explosive nephrolithopaxy: reality or fiction?

The use of silver and lead azide explosive charges for the percutaneous distintegration of renal calculi has been investigated. Charges of 10 mg or more reliably reduced calculi to fragments of extractable size; however, the concomitant tissue effects would preclude the use of such charges clinically. Smaller charges require multiple applications. High-speed flash photography demonstrated the unfocussed nature of these discharges. Considerable improvements are anticipated when the shock waves are focused. The use of Nonel tubing is described, and future developments are discussed.

Azides↗