Howard Sachs

VASOPRESSIN BIOSYNTHESIS—II INCORPORATION OF [35S]CYSTEINE INTO VASOPRESSIN AND PROTEIN ASSOCIATED WITH CELL FRACTIONS*

THE EST] TED rate of vasopressin biosynthesis in the dog is approximately 0.01 p g per hour (SHANNON, 1942). Despite this very low rate of synthesis, 35SS-labelled vasopressin, containing a total of 200-300 counts/min, has been isolated from dogs which had received about 1O1O counts/min [35S]cystine intravenously over periods of 16 to 36 hr (SACHS, 1960a). To investigate the intracellular path of vasopressin biosynthesis, greater incorporation had to be achieved than in the initial experiments (SACHS, 1960~). This was accomplished by means of the direct infusion of [T3]cysteine of high specific activity into the third ventricle of the brain of the dog

Studies on the intracellular distribution of vasopressin.

. By the use of such an approach it has been possible to carry out labelling experiments followed by the isolation of minute quantities (0.06 to 2.0 pg) of [35S]vasopressin from a number of intracellular compartments. The most highly labelled vasopressin molecules were associated with particulate material that sedimented in a relatively low centrifugal field. The cellular proteins showed labelling patterns different from that observed for vasopressin.

Cellular Processes Concerned with Vasopressin Biosynthesis, Storage and Release

ENDOCRINOLOGICAL and morphological studies (SCHARRER and SCHARRER, 1954) have suggested that the polypeptide hormone, vasopressin, is synthesized and held within neurosecretory particles (NSP)

VASOPRESSIN AND PROTEIN SYNTHESIS IN THE HYPOTHALAMUS OF THE DOG

in specific hypothalamic neurons, and that these NSP subsequently move via axonal flow into the neurohypophysis. In our laboratory, biochemical studies have been directed toward the localization of vasopressin within the neuron and the development of isotope methods for the isolation of minute quantities (0.06 to 2-0 pg) of labelled hormone from the NSP and other cellular organelles. A preliminary report (WEINSTEIN, MALAMED and SACHS, 1961) has described the isolation of NSP from the neurohypophysis of the dog. In the present paper are described experiments dealing with the isolation of NSP from the hypothalamo-median eminence (HME) complex of the dog, as well as studies concerned with the distribution of vasopressin among a number of cell organelles. In the following paper (SACHS, 1963) are reported the results of experiments dealing with the incorporation of [35S] cysteine into the vasopressin and protein molecules associated with the NSP and other cell structures.

EVIDENCE FOR A PRECURSOR IN VASOPRESSIN BIOSYNTHESIS.

The mammalian hypothalamo-neurohypophysial complex6 contains a group of well defined neurons whose cell bodies lie in the anterior hypothalamus and whose axons extend into the neurohypophysis; they are responsible for the elaboration of the polypeptide hormones, oxytocin and vasopressin. These neurons fulfill the morphological and functional criteria of neurosecretory cells. The most distinguishing morphological features of these neuron sare that their axon endings (in the neural lobe) about on blood vessel s rather than on other nerve cells, and that their axoplasm usually abounds with dense granules about O.1 to O.3 [i in diameter. These granules were presumed to contain the polypeptide hormones, oxytocin and vasopressin and therefore were termed “neurosecretory granules” (NSG) by morphologists. Since these NSG appeared to move in a proximo-distal direction, it was postulated that the synthesis of the hormones takes place in the perikaryon within the NSG, which then move in a protoplasmic flow along the axon to the region of the nerve endings. It is in this region that, in response to appropriate stimuli, the release of the NSG, or their contents, into the blood stream is thought to occur (2). The rudimentary features of this “neurosecretory process” are represented schematically in Fig. 1. Studies in our own and other laboratories have begun to outline some of the cellular mechanisms involved in the intermediate neurosecretory stages. In this manuscript, we report on some of our recent findings on the biosynthesis, storage, and release of vasopressin.

Biosynthesis and release of vasopressin and neurophysin.

Morphological and endocrinological studies have suggested that intracellular vasopressin is contained within neurosecretory particles (NSP) which are formed in the hypothalamus and move in a protoplasmic flow to the neurohypophysis where storage and release occur. I n the present studies, such NSP have been isolated from homogenates of the dog neurohypophysis by means of differential and density gradient centrifugation procedures (Sachs, 1 9 6 1 ~ ) . I n a similar manner, NSP from the dog hypothalamus have also been isolated. In addition, a number of other hypothalamic cell fractions were examined with respect to their content of pressor activity, protein, protein sulphur, DNA, RNA. and cytochrome oxidase. Subsequently, the specific activity of 35S-labelled vasopressin associated with the various cell fractions was determined after relatively short-term incorporation experiments. Extensive labelling of the vasopressin fractions was achieved by the use of highly labelled, [35S]-cysteine (prepared biosynthetically) which was infused directly into the third ventricle. Each labelled vasopressin fraction was purified to constant specific activity as previously described (Sachs 1961 b) . Several such experiments have indicated that: (1) there exist metabolically distinguishable intracellular pools of vasopressin; (2) the NSP are probably not the primary sites for hormone biosynthesis, since [35S]-vasopressin in the NSP always had the lowest specific activity: (3) vasopressin with the highest specific activity was always associated with a large granule fraction. This large granule fraction contained about 75 percent of the total cytochrome oxidase and 20 to 30 percent of the cytoplasmic RNA. Further fractionation with deoxycholate indicated that the more highly labelled vasopressin molecules were intimately associated with the membranous components rather than the ribosomes (which have been demonstrated as the primary sites for the synthesis of peptide bonds in protein in a number of animal, plant and bacterial systems). In the experiments described above, the incorporation of 35S-labelled cysteine sulphur into protein, was also studied. The proteins,