Conversion of Proparathyroid Hormone to Parathyroid Hormone by a Particulate Enzyme of the Parathyroid Gland*

The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathor-mone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution position during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH,-terminal region. Total homogenates and derived subcellular fractions (600 x g pellet, 5,000 x g pellet, 20,000 x g pellet, 190,000 x g pellet, and 190,000 x g supernatant) all catalyzed the conversion of exogenous [3H]- or [“Clprohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 x g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine chloroquine, by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [‘%]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in uiuo.

The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution position during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH,-terminal region. Total homogenates and derived subcellular fractions (600 x g pellet, 5,000 x g pellet, 20,000 x g pellet, 190,000 x g pellet, and 190,000 x g supernatant) all catalyzed the conversion of exogenous [3H]-or ["Clprohormone.
Over 60% of the converting activity was in the particulate fractions; the 190,000 x g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of ['%]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in uiuo.
Parathormone' is formed in vivo by proteolytic cleavage of its precursor, proparathormone (1, 2). The prohormone contains a basic hexapeptide on the NH, terminus (3) that precedes the 84.amino acid sequence of the hormone and may also contain a peptide segment on the COOH terminus which is as yet not fully characterized (4). The conversion of proparathormone to parathormone involves, therefore, the removal of both the NH,-terminal hexapeptide and the putative carboxyl adduct.
Virtually all of the hormone and prohormone of the parathyroid is associated with membrane-limited structures (5). Several lines of evidence indicate that newly formed proparathormone is transported through the cisternal space of the endoplasmic reticulum to the Golgi region of the cell where the conversion to parathormone takes place (6, 7). In addition, there may also occur along the same pathway complete *This work was supported in part by Grant AM 18323 from the National Institute of Arthritis, Metabolism and Digestive Diseases.
'The abbreviations used are: parathormone, parathyroid hormone; proparathormone, proparathyroid hormone; SHM buffer, 0.5 M sucrose, 0.02 M Hepes (N-Z-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0.001 M magnesium acetate; mRNA, messenger RNA; EGTA, ethylene glycol bi@aminoethyl ether)-N,iV-tetraacetic acid. degradation of the prohormone or hormone which may constitute a mechanism for the short or long term control of hormone production (8,9). Based on these considerations, it seems likely that the physiological enzymes responsible for the intracellular metabolism of the prohormone and hormone are also membrane-associated. Until now, however, little has been reported on the capacity of membranes of the parathyroid tissue to metabolize proparathormone or parathormone. In this report, we describe studies on the in vitro conversion of the prohormone to hormone catalyzed by a particulateassociated enzymatic converting system. This activity appears to represent that which forms the hormone from the prohormone in the intact cell.

Homogenates
of bovine parathyroid glands converted radioactive proparathormone substrate to a peptide which, based on its co-migration with authentic hormone during ion exchange chromatography, was tentatively identified as parathormone (Fig. 1). The production of the hormone was linear with time for up to 2 h, and corresponded to a decrease in the amount of the prohormone substrate. The converting activity was destroyed by incubation in a boiling water bath for 10 min.
In order to determine the subcellular distribution of the converting activity, experiments were performed in which tissue homogenates were separated by differential centrifugation into several fractions (  Automated sequence analysis was performed as described under "Experimental Procedure." The relevant portion of the sequence of bovine proparathormone is shown at the top of the figure in the one-letter code of Dayhoff (20). The serine (S) and leucine (L) residues in the sequence are underlined.
The sequence of proparathormone has been shifted 6 cycles to the left to account for the NH,-terminal hexapeptide.
dex G-100. It co-eluted exactly with authentic ['"Clparathormone, which indicated that their hydrodynamic radii were identical.
The partial amino acid sequence of the putative hormone product was examined by Edman degradation (Fig. 3) Based on these results, the failure to detect significant metabolism of parathormone in these experiments was not due to dilution of the radioactive substrate. In addition, the activity of the coverting enzyme between 1 and 3 h of incubation was demonstrated by the continuing conversion of proparathormone (Fig. 4). The initial loss of 10 to 12% of the hormonal substrate might have been due to adsorptive effects and produced no detectable metabolic products, even when higher levels of radioactivity were utilized. be isolated from the cell. Examples of these are proparathormone (1, 2), proalbumin (33)(34)(35), and proinsulin (24).