Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules.

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RESUMO

A method is described for the quantitative determination of the content of subcellular organelles such as secretory granules. Purified subcellular fractions of the organelle are prepared and aliquots are assayed for hormones, for example. To determine the number of organelles per fraction, known numbers of latex particles of a size similar to the organelle are added to other aliquots of the subcellular fractions. Latex particles and organelles are then pelleted together by centrifugation. The ratio between latex particles and organelles can be determined by morphometric analysis of ultrathin sections taken through the full thickness of the pellet. The number of organelles and hence their content of the substance assayed can then be calculated. We have applied this technique to posterior pituitary neurosecretory granules, the content of which has already been estimated by a different method. Newly formed neurosecretory granules from oxen and rats were found to have a content of approximately equal to 85,000 molecules of hormone and neurophysin. Aged neurosecretory granules from the same neural lobes appeared to contain less hormone and neurophysin, but this was shown to be the result of loss of material from the granules during isolation in media of 360 mosM. Such loss could be prevented by isolation in hypertonic (660 mosM) media.

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