Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

Unexpected oocyte growth after follicular antrum formation in four marsupial species.

During examination of maturing preovulatory marsupial oocytes we noted that oocyte diameters were invariably about 50% greater than the figures reported in earlier histological studies. As all previous investigations were limited to small follicles (at most 25% the size of the ovulating follicle), the present study was initiated to examine oocyte growth during the whole period of follicular development. Oocyte and follicle diameters were measured for three Australian (Trichosurus vulpecula, Macropus eugenii and Bettongia penicillata--fresh nonfixed material) and one American marsupial species (Monodelphis domestica--histological sections) in which multiple follicle development had been induced by exogenous gonadotrophin treatment. In all species oocytes were obtained from follicles ranging from pre-antral to immediately pre-ovulatory (maximum follicle sizes obtained were: T. vulpecula, 4.5 mm; M. eugenii, 4.3 mm; B. penicillata, 2.5 mm; M. domestica, 0.7 mm). In two of the species (T. vulpecula and B. penicillata) ovulated oocytes were also examined. In T. vulpecula and M. eugenii oocytes were found to achieve much greater diameters than previously reported from histological studies of small follicles (< 0.8 mm) and similar patterns of growth were found in the other two species. In the four species oocytes reached diameters about two to three times that found for eutherian mammals. It was concluded that the marsupial oocyte continued to grow after formation of the follicular antrum and that, although the rate of oocyte growth slowed in larger follicles, it continued into the period immediately before ovulation. In B. penicillata the largest oocytes were obtained after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that cortical granule formation is a periovulatory event in marsupials.

Formation of cortical granules was examined in superovulated oocytes from three marsupial species, brushtail possums (Trichosurus vulpecula) tammar wallabies (Macropus eugeniii) and grey short-tailed opossums (Monodelphis domestica) and in oocytes obtained during natural cycles in Macropus eugenii. Superovulation was induced by pregnant mares' serum gonadotrophin/gonadotrophin-releasing hormone (PMSG/GnRH) protocols and natural ovulation by removal of pouch young. Oocytes were collected after ovariectomy or by laparoscopically guided follicle aspiration into Hanks balanced salt solution (HBSS) supplemented with either 2.5% fetal calf serum (FCS) or 2.5% bovine serum albumin (BSA). Ovulated oocytes were collected by removing and flushing the oviducts with HBSS and fixed immediately for electron microscopy. There were no differences in the morphology or timing of formation of cortical granules between superovulated and naturally cycling animals. Cortical granules were absent from germinal vesicle (GV) stage follicular oocytes before the luteinizing hormone (LH) surge in all species. Dark cortical granules, similar in appearance to those seen in the oocytes of eutherian mammals, were found just beneath the plasma membrane (9 per 100 microns of plasma membrane) of preovulatory oocytes at germinal vesicle, metaphase 1 or anaphase 1 stages. In addition, they contained a number of less electron-dense cortical granules (12 per 100 microns plasma membrane). The cortical cytoplasm of preovulatory oocytes was rich in Golgi complexes actively involved in vesicle formation. Large numbers of dark cortical granules (90 per 100 microns plasma membrane) were found only in ovulated oocytes. A small number of cortical granules of lighter electron density were also present in ovulated oocytes. This suggests that the marsupial oocyte is following a very different timetable for cortical granule formation and accumulation from eutherian mammals and that oocytes of marsupials may not achieve cytoplasmic maturity until after ovulation. The significance of these events for fertilization and development remains to be established.     

A comparative study of chiasmata in male and femaleBettongia penicillata (Marsupialia)

The chiasma frequency and distribution does not differ substantially between the sexes in the marsupialBettongia penicillata. This result contrasts with the observations made on other species of marsupials.     

Cytotoxicity of glucose analogues in V79 multicell spheroids

Summary 2-Deoxy-d-glucose (2DG) and 5-thio-d-glucose (5TG) are glucose antimetabolites that are known to be selectively toxic to hypoxic cells grown as single cells or as monolayer cultures. These analogues were toxic to Chinese hamster V79 cells grown as multicell spheroids even under aerobic conditions. When spheroids, 500- to 600-μm diameter, were exposed to 7.5mm of these chemicals for 3 days, the number of clonogenic cells per spheroid dropped to 50% for 5-thio-d-glucose and 20% for 2-deoxy-d-glucose, relative to control values. Survivals were reduced to less than 1% when the experiment was repeated in glucose-free medium. Scanning electron photomicrographs of spheroids treated with 7.5mm of either analogue showed extensive damage to the outer cells. The cell killing observed was much more than could be predicted on the basis of the hypoxic fraction known to be present in these spheroids. The crowded tumor-like environment may make the cells vulnerable to the cytotoxic action of glucose analogues and other glycolytic inhibitors. Supported by the Ontario Cancer Treatment and Research Foundation, London Clinic.     

Biochemical and biophysical characteristics of diarrhea viruses of human and calf origin.

Polyacrylamide gel electrophoretic analysis of purified preparations of human and calf diarrhea viruses indicated eight polypeptide components, or possibly nine in the case of the calf diarrhea virus. Thermal denaturation and analytical studies of the calf diarrhea virus genome showed it to consist of 11 double-stranded segments of RNA. The placing of the human and calf diarrhea viruses together with other similar viruses into a genus separate from reovirus and orbivirus, but within the family Reoviridae, is discussed.     

Free N-acetylaminosugar in the seminal plasma of eutherian mammals.

The seminal plasma of seven eutherian species, including man, was examined for the presence of free N-acetylaminosugar. Only man had appreciable levels of N-acetylaminosugar in the semen (37-1 mg/100 ml), but in all cases this free N-acetylaminosugar was probably the result of breakdown of polysaccharides by semen glycosidases. High levels of free N-acetylaminosugar thus appear to be peculiar to marsupials.