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pH-Dependent Stimulation of Peptidylglycine -Amidating Monooxygenase Activity by a Granule-Associated Factor^*

Department of Neuroscience, The Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Address requests for reprints to: Dr. Betty A. Eipper, WBSB 907, Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

Peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3) is a granule-associated enzyme that catalyzes the production of a-amidated peptides from their glycineextended precursors, a posttranslational modification often required for full biological activity. PAM activity in crude homogenates of bovine neurointermediate pituitary has an acidic pH optimum for the peptide substrate -N-Ac-Tyr-Val-Gly. During purification, the pH optimum shifts, so that purified bovine (b)PAM exhibits an alkaline pH optimum for this substrate with virtually no activity below pH 6.5. A factor that restores the ability of purified bPAM to produce a-amidated products at pH 6 was identified. In rat anterior pituitary this factor (denoted SPAM for stimulator of PAM activity) was a soluble protein with a mol wt of 44 K by gel filtration; its stimulatory activity could be reduced or eliminated by trypsin digestion or boiling. SPAM stimulated PAM activity at acidic pH by increasing the apparent V_max and decreasing the apparent Michaelis-Menten constant (K_m) for the peptide substrate. Like PAM, SPAM activity is localized to the secretory granule. Levels of SPAM activity in various rat tissues correlated closely with levels of PAM activity, with the greatest amount of SPAM activity in atrium, anterior pituitary, and neurointermediate pituitary. The distribution of PAM and SPAM between soluble and membrane fractions also correlated closely. In AtT-20 cell lines transfected with a complementary DNA (cDNA) encoding the full-length bPAM precursor, both SPAM and PAM activities were increased compared to wild type cells; both activities were decreased in a cell line expressing an antisense rat (r)PAM mRNA. In marked contrast, an AtT-20 cell line transfected with a cDNA encoding a truncated, soluble form of bPAM had elevated levels of PAM activity, but levels of SPAM activity were not increased compared to wild-type cells. These results suggest that SPAM activity is closely linked to the expression of full-length PAM. The interaction of PAM and SPAM may represent a site for regulation of the synthesis of bioactive peptides, particularly at low intragranular pH. (Endocrinology 127: 2771–2778,1990)

Footnotes

^* This work was supported by NIH Grants DK-32948 and DK-32949 and NIDA Grants DA-00097 and DA-00098. Portions of this work were presented as Abstract 1313 at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990.

Received July 6, 1990.

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