Hydrolytic enzymes in the central vacuole of plant cells
The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: ..cap alpha..-mannosidase, ..beta..-N-acetylglucosaminidase, ..beta..-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter (24% sucrose, w/w).
- Research Organization:
- Michigan State Univ., East Lansing, MI (United States)
- DOE Contract Number:
- EY-76-C-02-1338
- OSTI ID:
- 5410195
- Journal Information:
- Plant Physiol.; (United States), Vol. 63:6
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PLANT CELLS
HYDROLASES
CELL WALL
CENTRIFUGATION
CHOLINE
ENZYMES
ESTERASES
GLYCOSYL HYDROLASES
NICOTIANA
PEPTIDE HYDROLASES
PHOSPHATASES
PLANT TISSUES
PURIFICATION
TISSUE CULTURES
TRITIUM
ALCOHOLS
AMINES
AMMONIUM COMPOUNDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
HYDROGEN ISOTOPES
HYDROXY COMPOUNDS
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLANTS
QUATERNARY COMPOUNDS
RADIOISOTOPES
SEPARATION PROCESSES
YEARS LIVING RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques