Two isoforms of creatine kinase (CK, E.C. 2.7.3.2), the brain type BB-CK and the mitochondrial-bound MiMi-CK, as well as adenylate kinase (myokinase, E.C. 2.7.4.3) were identified in washed spermatozoa from chicken and man by cellulose polyacetate electrophoresis and immunoblots. BB-CK was localized by indirect immunofluorescence staining within the sperm tail but not in the head portion. MiMi-CK is confined to the midpiece region rich in mitochondria and has been localized directly by immunogold staining within the mitochondria. In contrast to chicken, seminal plasma from man was also found to contain considerable amounts of BB-CK. Total creatine content of spermatozoa (8-15 mM) and seminal plasma (3.8 +/- 0.4 mM) as well as preliminary experiments with metabolic blockers indicate a dependence of sperm motility on CK and phosphoryl creatine (CP). The presence of two CK isoforms located in different 'compartments' of spermatozoa suggests a CP-shuttle in sperm similar to that described for cross-striated muscle.
Four different types of endo-fl-l,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the eoll gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The eoll gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or A D H 1 locus of the yeast. Integration into the PGK1 or A D H 1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the eoH gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of fl-glucans in worts.
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