Identification of a region of mouse zona pellucida glycoprotein mZP3 that possesses sperm receptor activity.

The ability of mouse zona pellucida glycoprotein ZP3 (mZP3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 108, 1-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, approximately 55,000 apparent M(r), that exhibited sperm receptor activity in vitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function.     

Tiron as a spin-trap for superoxide radicals produced by the respiratory chain of submitochondrial particles

Tiron can be used as a spin-trap for O2 radicals generated by the respiratory chain of submitochondrial particles (SMP). Using this sensitive method, it was shown that the O2 (radical) production by the succinate-oxidizing SMP can be reduced by antimycin or 4-nonyl-2-hydroxyquinoline-N-oxide, the effects of both antibiotics being abolished and prevented by cyanide. It is suggested tht the O2 radicals are produced due to autooxidation of ubisemiquinone which is formed as an intermediate upon one-electron oxidation of CoQH2 by cytochrome c1. The effects of antimycin, 2-nonyl-4-hydroxyquinoline-N-oxide and cyanide on the O2 (radical) generation correlate with the effects of these inhibitors on a steady-state concentration of ubisemiquinone predicted by the Mitchell's Q-cycle hypothesis.     

Effect of a high-protein diet on the gene expression of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.