Preliminary Evidence for a Microsomal Precursor to Human Parathyroid Hormone

Abstract

Human parathyroid glands from a patient with chronic renal failure were used to study in vitro biosynthesis of immunoreactive parathyroid hormone. Parathyroid glands were incubated in the presence of [3Hlleu-cine, and classical subcellular fractions were prepared. Peptides from acetic acid extractions of the fractions were separated on Sephadex G-75 Superfine. Eluates were assayed for immunoreactive parathyroid hormone by a double-antibody technique. Leucine was incorporated into several large immunoreactive peptides of parathyroid hormone having estimated molecular weights of >25,000, 18,000, and 13,000. The large forms of immunoreactive parathyroid hormone were prominant in the microsomes. The secretion granule fraction, as well as the patient's serum, contained principally immunoreactive parathyroid hormone with an apparent molecular weight of 10,000.We have previously demonstrated the existence of immunoreactive peptides of human parathyroid hormone (PTH) that have apparent molecular weights larger than that of highly purified bovine PTH, which has a molecular weight of 9500 (1). Furthermore, we have found in studying the subcellular fractions of human parathyroid tissue that the immunoreactive peptides larger than purified bovine PTH can be localized to the microsome fraction of the gland, which contains protein synthetic components of the cell. From this information we have postulated that human PTH is synthesized by way of a large precursor molecule that is subsequently cleaved to a peptide with molecular weight of about 10,000 and which may be the principal form of PTH stored in the parathyroid's secretory granules. To test this hypothesis, we have incubated human parathyroid tissue with [8H]-leucine to study the in vitro synthesis of this possible precursor to human PTH. METHODSIn Vitro Incorporation of [8HjLeucine. Human parathyroid tissue was obtained fresh at the time of subtotal parathyroidectomy on a patient with chronic renal failure. The tissue was kept briefly on ice until dissected free of connective tissue and minced into pieces of about 1 mm. The tissue was incubated in 5 ml of incubation medium which was Kreb's bicarbonate buffer (gas phase 95% 02-5% C02), containing 1.3 mM calcium, 1.1 mM magnesium, 15 mg/liter of each of 16 amino acids without leucine, 100 ,Ci of [3H]leucine and 1% bovine-serum albumin. The incubation vessel was gassed with 95% 02-5% CO2 and then placed into a shaking water bath at 37°. After 30 min, the vessel was again gassed and Abbreviation: PTH, parathyroid hormone. Pellet from 10,000 X g for 20 min and mitochondrial fraction) F III (microsome fraction) Pellet from 100,000 X g for 60 min F IV Residual soluble fraction incubated for an additional 30 min. After 60 min of incubation, the incubation medium was removed and the tissue was washed twice briefly with ice-cold 0.25 M sucrose. The tissue was homogenized in 5 ml of ice-cold 0.25 M sucrose. A 1-ml aliquot of the homogenate was removed; the remaining suspension was carried through differential centrifugatio...

Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

“…Wong and Lindall (29) and Benson et al (30) recently reported findings that are consistent with the production and secretion of immunoreactive molecular forms larger than PTH.…”

Pre-proparathyroid hormone: Analysis of radioactive tryptic peptides and amino acid sequence

“…The absence of a methionine residue (the initial residue believed to be involved in translation in eukaryotic systems (see Kemper et al, 1976)) preceding the amino terminus of ProPTH makes it extremely unlikely that initiation of polypeptide chain growth occurs with the synthesis of a molecule as small as ProPTH. On the other hand, it also is possible that precursors of PTH larger than Pre-ProPTH exist that are synthesized and secreted into the circulation under abnormal conditions (Wong and Lindall, 1973;Benson et al, 1974).…”

Pre-proparathyroid Hormone: A Direct Translation Product of Parathyroid Messenger RNA

“…It seems possible that under unusual circumstances pre-ProPTH may not be cleaved and may accumulate in the cell or even be secreted. Several large proteins, immunoreactive to PTH antisera, have been found in hyperplastic parathyroid tissue (23) and in blood of patients with ectopic hyperparathyroidism (24). Pre-ProPTH may correspond to one of the immunoreactive species noted in these studies.…”