Heavy meromyosin subfragment-1 and its trinitrophenylated derivative have been chromatographed on immobilized ATP, ADP and adenosine 5'-(geta, gamma-imino) triphosphate affinity chromatography ...columns, in the presence and in the absence of Ng-2+ or Ca-2+.ma-32-P ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5' (beta, gamma-imino) triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the Pi-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.
Thermophilic ethanol fermentations are of interest to industrial alcohol production because both the pentose and hexose fraction of biomass can be directly fermented in high yield (i.e., mol ...ethanol/mol substrate consumed), and because of potential novel process features associated with high temperature operation. As a net result, the co-culture cellulose fermentations described here may have the potential to convert more substrate to alcohol than some other bioconversion systems described see Figure 11, (2). However, considerably more fundamental and applied research is required before realistic economic assessments can be made. Detailed analysis of the data presented above suggests key control parameters for thermophilic ethanol production (see Table IX). Understanding in detail the physiological and biochemical features that control rate limitation, yield limitation and concentration limitation appears to me as trends for future applied and fundamental studies on thermophilic ethanologenic bacteria. It is worth noting from the data reviewed here that understanding control of any one of these 3 major limitations is complex and multi-faceted. Indeed, improvement of ethanol tolerance (i.e. the ability to produce greater than 1% ethanol at high rates) in these bacteria appears to involve challenges by all three limitations. Furthermore, the biochemical basis for alcohol tolerance in thermophilic ethanologens appears to vary in different species. For example, the ethanol dehydrogenase of C. thermocellum is inhibited by physiological concentrations of alcohol (i.e. 1%) whereas, the reversible activity of T. brockii or C. thermohydrosulfuricum enzyme is increased by higher solvent concentration (greater than 5%).
Separation of heavy meromyosin subfragment-1 treated with N-ethyl maleimide (MalNEt) into native -SH1- and -(SH1, SH2)-blocked protein populations could be achieved by affinity chromatography on ...agarose-ATP columns in the presence of Mg2+ or Ca2+. Covalent bridging of the two -SH groups by p-phenylenedimaleimide gave a product which has the same affinity of binding to ATP columns as the doubly blocked MalNEt preparation. Treatment with p-phenylenedimaleimide abolished binding to immobilized F-actin columns, whereas modifications by MalNEt did not affect adsorption by this chromatographic medium. Affinity chromatography on immobilized nucleotide and actin columns is suggested as an analytical tool in the study of the involvement of thiol groups in the myosin active site and its conformation.
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