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SUMMARYThe appearance and the yield of sedimentable organelles from the cotyledons of Phaseolus vulgaris L. were measured with respect to the influence of the method of homogenization, the choice of dry or imbibed seeds, and the composition and the concentration of the homogenization medium. Sucrose, (0.2-0.8 M), the addition of Mg^"" (2 X 10~^M) or EDTA (3 X 10~^M) or the replacement of sucrose with mannitol made little difference to the average size distribution or the amount recovered of the 21 000 ^^v ^ 10 min pelleted organelles, the main protein body-containing fraction of the homogenate. Slicing the imbibed seeds with a razor blade or by a mechanical rotary cutter combined with a short extraction was the gentlest and the most efficient method for the production of organelles. Cell homogenates were fractionated on the basis of size by differential sedimentation into several subcellular fractions. These included a residue, starch particles (300 ^ X 10 min), a main protein body fraction (21 000 ^^v X 10 min), 38 000 ^av^ 60 min pellets, 78 000 ^av X 240 min pellets and a final supernatant. The activity in these fractions of several catabolic enzymes was measured. Most of the activity of the acidic nucleases, amylases, the BAPA-ase and a substantial part of the acidic phosphatase and the cathepsin A-type enzyme(s) were found in the supernatant, while no specific DNA-ase was found in these seeds. Protein bodies contained some of the trypsin inhibitors and most (if not all) of the lectins and Glycoprotein II. Most of the endopeptidase (azoproteinase) activity of the cotyledons appeared to be tightly bound to the cell walls or to other insoluble structural elements. Protein bodies and other intracellular organelles had very little autolytic activity and, thus, the main storage proteins of the seed were not degraded during the first 4 days of germination.

1986. Intracellular localizatioD of some peptidases and a-mannosidase in cotyledons of resting kidney bean, Fhaseolus vulgaris. -Physiol. Plant. 68: 75-^.Cotyledoiis of resting kidney beans {Phaseolus vtdgaris, L., cv. "Processor") contain high activities of two alkaline peptidases, an aminopeptidase (EC 3.4.11) acting on Leu-Tyr and Leu-Gly-Giy and a dipeptidase (EC 3.4.13) hydrolysing Ala-Gly together with low activities of neutral naphthyiamidases (marker substrate Leu-^-NA) and of add carboxypeptidases (EC 3.4.16; marker substrate Z-Phe-Ala). The intraeellular localisation of these peptidases and that of a-mannosidase (EC 3.2.1.24) was studied by subceliuiar fractionations in different media. In density gradient centdfugations in non-aqueous giycerol-potassium iodide media the alkaline peptidases remained mainiy in the application zone suggesting localisation in the cytosol. The carboxypeptidase and a-mannosidase activities banded mainly in the protein body zone, but about 15-30% of each activity was found in the cell wall zone. Results obtained by short centrifugation in glycerol or high-density sucrose solutions (65/70%) and by the isolation of essentially pure cell wall fractions confirmed these assignments. The results are in accordance with previous suggestions that the abundant alkaline peptidases may play a role in the mobilization of reserve proteins in germinated seeds by hydrolysing peptides which are produced initially within the protein bodies by acid proteinases and carboxypeptidases and which subsequently leak out or are transported from the autolyzing protein bodies to the cytosol.Additional key words -Carboxypeptidase, dipeptidase, leucine aminopeptidase, naphthylamidase, subceliuiar fractionation. A. Mikkonen (reprint requests),

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