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Pregrowth Hormone: Evidence for Conversion to Growth Hormone During Synthesis on Membrane-Bound Polysomes

Department of Biological Sciences, Columbia University New York, New York 10027

^* To whom all correspondence should be addressed. Present address: Sloan-Kettering Institute for Cancer Research, New York, New York 10021.

Abstract

Previous investigations have shown that the product of the translation in vitro of messenger RNA coding for growth hormone is a protein larger than growth hormone, designated pregrowth hormone. It was suggested that pregrowth hormone is a biosynthetic precursor of growth hormone. In the present studies we have investigated further the metabolic relationship between growth hormone and pregrowth hormone. Polysomes isolated from a clonal strain of rat pituitary (GC) cells were incubated in the presence of S-100 extracts of mouse Krebs ascites cells. Incubation of membrane-released or free polysomes in extracts containing [³H]leucine demonstrated that growth hormone is synthesized by membrane-bound polysomes. Incubation of membrane- released polysomes, isolated from GC cells labeled briefly with [³H]leucine, in extracts containing only unlabeled amino acids demonstrated that the polysomes contained nascent pregrowth hormone at the time of their isolation, and thus that intact pituitary cells are engaged in the synthesis of this protein. The observation of labeled growth hormone in this experiment suggested that pregrowth hormone is cleaved to growth hormone during synthesis. To distinguish between alternative interpretations of this experiment, a kinetic experiment was performed in which [³H]- leucine-labeled membrane-released polysomes were incubated in unlabeled extracts, and the radioactivity in pregrowth hormone and growth hormone measured at various times. The observation that radioactivity in pregrowth hormone was still increasing when radioactivity in growth hormone had plateaued showed that pregrowth hormone is cleaved to growth hormone during synthesis in pituitary cells. Furthermore, the observation of radioactivity in pregrowth hormone at the earliest times examined provided strong evidence that cleavage of preGH to GH occurs when nascent preGH chains are at various stages of completion.

Footnotes

Supported by Grants GM-21000 from the National Institutes of Health and PCM 74-19373 from the National Science Foundation.

Received February 24, 1977.