NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1 M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present.
LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae). The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised. However, the antibody failed to recognise the predominant protein present after heating the extract at 80 degrees C for 10 min. This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 degrees C for 10 min. Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 degrees C compared with growth at 30 degrees C. However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol.
The cleavage stage (CS) H1, H2A, and H2B histones of the sea urchin, which have previously been identified by their distinct electrophoretic mobility on Tritonlacidurea gels, are known to be maternally expressed during oogenesis and have been implicated in chromatin remodeling of the male pronucleus following fertilization. Here, we describe the isolation of these three CS histones by reverse-phase HPLC chromatography. Moreover, a novel CS H3 protein was identified by the same purification procedure. A low incorporation of radioactive amino acids into the CS hiqtones during early development revealed that the bulk of these proteins in the blastula embryo are derived from the maternal pool of the egg. Amino acid analysis, together with the previously described electrophoretic mobilities, unequivocally identified the purified proteins as CS histones. Peptide sequence analysis confirmed the novel nature of the CS variants as they are distantly related to the early, late, and sperm histone subtypes of the sea urchin. The CS H1 protein displays highest sequence similarity with the H1M (B4) histone of Xenopus laevis, indicating that the frog H1M protein may be a vertebrate homologue of the CS HI histone. These data suggest an ancient evolutionary origin and wide distribution of the CS histone variants.
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