Characterization, targeting, and functional analyses of male germ cell-specific type 1 hexokinases in the mouse [electronic resource].

Travis, Alexander John Anthony.
218 p.
Veterinary medicine.
Local subjects:
Penn dissertations -- Cell and molecular biology. (search)
Cell and molecular biology -- Penn dissertations. (search)
System Details:
Mode of access: World Wide Web.
Three germ cell-specific isoforms of type 1 hexokinase, (HK1) are transcribed during spermatogenesis in the mouse: HK1-sa, HK1-sb, and HK1-sc. Each transcript encodes a germ cell-specific sequence that replaces the targeting domain of somatic HK1. A population of HK1 in murine sperm has biochemical characteristics differing from somatic HK1. This protein had been purported to be a sperm-surface ZP3 receptor.
Aims. (1) to define the HK1 isoforms present in male germ cells, and characterize their subcellular localizations. (2) To delimit domains involved in targeting to specific organelles. (3) To investigate functions of this protein in sperm, and regulation of its activity during sperm capacitation.
Methods. Anti-peptide antisera were generated to distinguish among the HK1 isoforms. Recombinant proteins were generated to serve as positive controls. Immunoblot analysis was used to identify the isoform(s) present. Immunoelectron microscopy was used to localize the protein in spermatozoa. Targeting analysis was performed using a heterologous system to express constructs containing different domains of the germ cell-specific transcripts linked to green fluorescent protein. Measurements and localization of enzyme activity were performed by spectrophotometric and histochemical methods. Function as a ZP3-binding protein was investigated using binding assays.
Results. HK1-SC is the only HK1 isoform found in spermatozoa and developing male germ cells. It is found associated with membranes of the head, mitochondria of the midpiece, and the fibrous sheath of the flagellum. The germ cell-specific domain targets fusion proteins to the endoplasmic reticulum and plasma membrane by means of a novel, non-hydrophobic motif. HK1-SC does not bind ZP3, and is not regulated with capacitation. Endogenous reducing power is regulated with capacitation, defining the first biochemical function of this maturational process.
Conclusions. The glycolytic pathway is most likely arrayed down the length of the fibrous sheath, poised to deliver ATP to the axoneme. HK1-SC targets to membranes by means of a novel motif. Pentose phosphate pathway activity is most likely regulated during sperm capacitation.
Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 1999.
Source: Dissertation Abstracts International, Volume: 60-07, Section: B, page: 3068.
Adviser: Gregory S. Kopf.
Local notes:
School code: 0175.
Kopf, Gregory S., advisor
University of Pennsylvania.
Contained In:
Dissertation Abstracts International 60-07B.
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Restricted for use by site license.
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