Subcellular fractionation by centrifugation of homogenates of the neural lobe of the bovine pituitary gland: identification of different pools of hormone in the homogenate and isolation of neurosecretosomes.
Homogenates of bovine pituitary neural lobe tissue were subjected to differential centrifugation. Six subcellular fractions (I-VI) were obtained and the distribution among them of various cell organelles was studied by means of markers. Fraction II (800-3000 gav), the nerve-ending fraction (neurosecretosomes), contained sedimentable lactate dehydrogenase, hormone and a large proportion of the total Mg2 + +Na+ +K+-ATPase. 2. Lysis of the neurosecretosomes in hypotonic sucrose solutions led to loss of lactate dehydrogenase and vasopressin. 3. Centrifugation of the granule fraction (IV) on a sucrose gradient (1.3-2.0 M sucrose) gave a bimodal distribution of vasopressin. Purified neurosecretory granules were recovered from the denser band. Centrifugation on modified gradients (0.8-2.0 or 0.4-2.0 M sucrose) increased the yield of hormone in the denser band but the purity of the granules was decreased. 4. Considerable purification of the neurosecretosomes (fraction II) was achieved by "washing". Centrifugation of washed neurosecretosomes on sucrose density gradients led to the accumulation of all activities at a region between 1.4 and 1.5 M sucrose. 5. The distribution of Mg2+ +Na+ +K+-ATPase in centrifugal fractions indicated that the neurosecretosomes had been isolated in relatively high yield.
