[New technologies in the prosthetic management after amputations]. / Neue Techniken in der prothetischen Versorgung nach Amputationen.

BACKGROUND: In Germany around 70,000 amputations are carried out on extremities each year. Modern prosthetic functional components have become more and more sophisticated and must be understood and applied by their users to be of beneficial use in everyday life. The prosthetic socket is the most important component of modern extremity prosthetics. OBJECTIVES: Which demands have to be met by a modern prosthetic socket so that innovative function-improving components in prosthetics can be successfully applied? MATERIALS AND METHODS: Complex prosthetic technologies are rarely compatible with a lower overall weight of the prosthesis. The increase in functionality also produces differentiated force effects on the human body. Modern socket technologies, therefore, have to compensate for the increased strain and counteract the increasing dynamics between the stump and the prosthesis. This can be achieved through the application of adhesive socket materials and through new adhesive mechanisms. Form variants can also improve the connection between stump and prosthetic socket. RESULTS: The improvements in prosthetic socket technology presented here have a lasting positive effect on the daily routine of many amputees. Not only do they improve the control and application of modern prosthetic components, but also clearly enhance the wearing comfort. CONCLUSION: The prosthetic socket is crucial for the success of exoskeletal prosthetic management. The better we succeed in making the human body and the prosthetic socket an entity, the more usefully and comfortably innovative prosthetic methods can be applied.

Amino acid sequence of a peptide containing an essential cysteine residue of pig heart aconitase.

Pig heart aconitase reacts with one mole of phenacyl bromide per molecule to give complete inactivation due to the alkylation of a cysteine reside at the active site. A tryptic peptide containing this essential residue has been isolated and its amino acid sequence determined at Ile-Gln-Leu-Leu-Cys *-Pro-Leu-Leu-Asn-Gln-Phe-Asp-Lys by manual methods and by the use of an automated solid phase sequencer. There is a limited similarity in amino acid sequence between this peptide and other peptides containing the cysteine residues involved in the binding of the iron-sulfur clusters of high-potential iron-sulfur protein of Rhodopseudomonas gelatinosa and rubredoxins from various bacteria.

Analysis of the iron-sulfur cluster of aconitase by natural and magnetic circular dichroism.

We have examined the iron-sulfur cluster of aconitase, a high-potential iron-sulfur protein, by absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of unactivated aconitase, which is presumably oxidized, is similar to those of reduced two iron-two sulfide ferredoxins but distinct from the MCD of known four iron-four sulfide proteins. The magnitude of the natural CD of unactivated aconitase also suggests the absence of four iron-four sulfur clusters. Reduction of the enzyme with dithionite and activation with the cysteine-ascorbate-ferrous ion activation mixture generate spectra which are significantly different from those of any iron-sulfur protein seen to date. We interpret these results as indicating that aconitase does not contain a four iron-four sulfur cluster generally thought to be characteristic of high-potential iron-sulfur proteins. It could contain a two iron-two sulfur center or some other center such as a cyclic three iron-three sulfur center.

Structural basis for aconitase activity inactivation by butanedione and binding of substrates and inhibitors.

Aconitase (citrate(isocitrate)hydro-lyase, EC 4.2.1.3) prior to activation demonstrates a single binding site for substrates and inhibitors. On the basis of kinetic experiments, at pH 8.5 and 37 degrees C, with monomeric butanedione in borate, this binding site was found to contain a single arginine residue. Dissociation constants at pH 8.5 and 37 degrees C, determined from inhibitory effects on butanedione inactivation rates are: citrate, 0.74 mM; D-isocitrate, 0.33 mM: cis-aconitate, 0.52 mM; tricarballytate, 0.42 mM; trans-aconitate, 0.025 mM. Corresponding dissociation constants for the active enzyme are: tricarballylate, 0.39 mM; trans-aconitate, 0.14 mM. Active site Fe2+ added to the enzyme on activation is therefore not required for binding. Km values are: citrate, 0.23 mM and cis-aconitate 0.012 mM. Binding to active enzyme is considered to be transition state binding.

Properties of pig heart aconitase.

Comparison of pig heart aconitase (Kennedy et al., 1972) with yeast (Candida lipolytica) aconitase (Suzuki et al., 1973) reveals similarities in molecular weight and iron content but not in sulphide content. Comparison with the Mildvan & Villafranca (1971) pig heart aconitase preparation reveals differences in iron ligands, specific activity and other properties; these differences possibly arise from protein association as pig heart protein associates under a variety of conditions. The electron spin resonance spectrum, g 4.25, and the low molar relaxivity, 473m(-1).s(-1), of water H(+) suggest the presence of high-spin Fe(III) unco-ordinated to water in the enzyme. The iron chromophore on acid titration at 320nm gives a curve with an inflexion at pH4.2. Ten of 16 expected thiol equivalents are titrated with p-hydroxymercuribenzoate suggesting the presence of cystine as well as cysteine residues. Inhibition of the activation of inactive (activatable) enzyme is sigmoidally related to the molar ratio, p-hydroxymercuribenzoate/enzyme with 10-11mol of mercurial compound causing complete inhibition. Active enzyme, free from activating reagents, requires high molar ratios of mercurial compound for rapid inhibition. In terms of p-hydroxymercuribenzoate the enzyme then lacks an essential thiol group.

Iron and aconitase activity.

Aconitase activated with Fe(2+), cysteine and ascorbate incorporates 1 g-atom of Fe(2+)/mol. Loss of this Fe(2+) by transfer to ferrozine, a Fe(2+) chelator, results in loss of activity. Ascorbate increases the rate of transfer of the essential Fe(2+) whereas citrate retards the rate of transfer. Transfer of Fe(2+) from inactive aconitase, 2 g-atoms of Fe/mol, can be accomplished in the presence of urea and ascorbate. The correlation of activity with the presence of an added g-atom of Fe(2+)/mol leads to the conclusion that active aconitase has only one active site per mol.