Lawson L. Rosenberg

Presence and distribution of gonadotropin and thyrotropin in the pars distalis of the lizard Anolis carolinensis

Abstract Partial hypophysectomy and bioassay of pituitary extracts were used to study the presence and distribution of thyrotropin (TSH) and gonadotropin within the pars distalis of the adult male lizard ( Anolis carolinensis ). Removal of different regions of the pars distalis indicated that gonadotropin was produced throughout the gland, whereas TSH was produced primarily by the caudal region of the gland. Injections of extracts of either rostral or caudal halves of the pars distalis stimulated testicular growth and interstitial cell activity in hypophysectomized Anolis , and the concentration of gonadotropin appears to be approximately equal in the two regions. The existence of two separate gonadotropins in reptiles is questioned. Hypophysectomy produced a gradual decline in the rate of radioiodine uptake by the thyroid; 24-hr uptake values 11 days after hypophysectomy were only 20% of control values, and after 21 days they were only about 2% of controls. A reduced rate of urinary 131 I excretion also occurred in hypophysectomized animals. Chromatographic analysis of hydrolyzed thyroid homogenates and plasma indicated a relatively high rate of T 4 production and secretion by the thyroids of intact animals, suggesting that the reptilian thyroid is not as sluggish as previously described. Hypophysectomy significantly reduced the rate of thyroid hormone biosynthesis. Only the caudal half of the pars distalis appeared to have TSH activity as judged by the radioiodine uptake and labeling of T 4 in hypophysectomized Anolis injected with pituitary extracts. Animals injected with caudal tissue also showed significantly more lean growth than the other hypophysectomized animals. These results are discussed in relation to histological studies of pituitary cell types. Previous identification of pituitary thyrotropes and gonadotropes appear incorrect and a tentative scheme for the functional identification of the 5 recognized cell types in the pars distalis is proposed in light of recent bioassays of the pituitary hormones in reptiles.

Studies on a cyclic AMP-dependent protein kinase from rat thyroid gland.

Abstract We have confirmed the presence of cyclic AMP-dependent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) in the soluble protein fraction of rat thyroid homogenates, and have purified the enzyme with a 20% yield by gel filtration on Sephadex G-200 followed by ion exchange chromatography on DEAE-cellulose. Kinase activity was assayed in a standard system with [γ-32P]ATP and a mixed fraction of calf thymus histones at pH 6.5, 30°C, in the presence and absence of cyclic AMP. Two peaks of cyclic AMP-dependent protein kinase activity were resolved by gel filtration and each was further purified on DEAE-cellulose and designated DKI and DKII: specific activities were, respectively, 5171 and 2080 units/mg protein, representing an approximate 700-fold purification of activity based on assay of the crude extracts. However, crude thyroid extracts reveal only 1/10 of their inherent kinase activity, probably due to the presence of ATPase activity, and in addition, to interaction of the large amounts of thyroglobulin with the histone phosphoryl acceptor. A unit of activity is defined as 1 pmol phosphate transferred from phosphoryl donor to acceptor per min. The two kinases were stable for up to 1 month when stored at 4°C. The Km with the respect to cyclic AMP is 6.1 · 10−8 M for DKI and 3.1 · 10−8 M for DKII, and with respect to ATP, in the presence and absence of cyclic AMP (1 · 10−6 M), respectively, is 1.0 · 10−5 M and 1.2 · 10−5 M for DKI, and 0.93 · 10−5 M and 0.75 · 10−5 M for DKII. These values are similar to those reported for cyclic nucleotide dependent protein kinases from other tissues. Molecular weights estimated by gel filtration are 230 000 and 152 000 for DKI and DKII respectively. Sedimentation coefficients of 8.7 for DKI and 6.6 for DKII were estimated by centrifugation of the material in the linear sucrose density gradients. Enzymatic profiles of the gradients of the heavier kinase consistently showed a small peak corresponding to 6.6 S. Although the data might suggest that multiple forms of protein kinase exist in the rat thyroid, they are also consistent with the presence of a kinase in several states of aggregation peculiar to the purification procedure. Both DKI and DKII when incubated with cyclic AMP were converted to 4.8 S material that was essentially completely nucleotide independent, consistent with the well-described disaggregation of the catalytic subunit(s) from protein kinase holoenzyme.