We describe the full-length (72 kDa) myotonin protein kinase (Mt-PK) and demonstrate its kinase activity. The 72-kDa protein corresponds to the translation product from the first in-frame AUG codon. Ser residue and phosphorylates the synthetic peptide Gly-ArgGly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in the phosphorylation site. We examined phosphorylation of the voltage-dependent Ca2+ release channel, or dihydropyridine receptor (DHPR), by recombinant Mt-PK. We observed that the f3 subunit of DHPR was phosphorylated in vitro by Mt-PK. A a8-subunit DHPR peptide containing some of the Ser residues predicted to be phosphorylated was synthesized and found to be a substrate for Mt-PK in vitro. We conclude that the 72-kDa Mt-PK has a protein kinase activity specific for Ser residues.

No abstract

Prolactin binding sites have been demonstrated previously in rat choroid plexus using in vivo radioautography (Walsh et al. 1978). In the present study we have employed this procedure to characterize further the binding specificity of these sites. Following the injection of 125I-hGH or 125I-oPRL an intense radioautographic reaction was observed over the choroid plexus. The reaction was significantly reduced by coinjecting excess unlabeled hGH or oPRL but not bGH. The specific binding of 125I-oPRL to choroid plexus from rat, rabbit, sheep and pig was demonstrated by in vitro assays. Subsequently a survey of 125I-oPRL specific binding in a number of regions of pig brain indicated that the highest binding was in choroid plexus. A detailed study of the characteristics of 125I-oPRL binding to pig choroid plexus was undertaken. Specific binding increased with choroid plexus homogenate protein to a maximum of 30% (3.0 mg protein/tube). Binding was maximum at 4 degrees C by 30-40 h of incubation. During the incubation the integrity of 125I-oPRL in the incubation medium declined steadily to 50% after 20 h and 35-40% after 48 h. Radioactivity eluted from binding sites was fully intact as judged by rebinding to lactogen receptor-enriched membranes. Binding showed a broad pH optimum of 5.5-7.5. On cell fractionation of choroid plexus binding sites were enriched in microsomes. The binding of 125I-oPRL and 125I-hGH was inhibited in a dose-dependent manner by unlabeled lactogens and was of high affinity. hGH and oPRL were equipotent inhibitors of the binding of both radioligands whereas bGH and a variety of structurally unrelated peptides were non-inhibitory.

In previous studies we have shown that 125I-labeled prolactin is taken up by a receptor-dependent process and concentrated in an intact form in Golgi elements from female rat liver (J. Biol. Chem., 1979, 254:209-214). In this study we have examined the effect of colchicine on this uptake process into Golgi elements. Colchicine [25 mumol (10 mg)/100 gm body wt] was injected intraperitoneally in adult female rats, and hepatic Golgi fractions were prepared at 1, 2, and 3 h postinjection. The enzyme recoveries and morphological appearance of fractions from colchicine-treated and control (alcohol alone) animals were similar. At times greater than 1 h after colchicine there was a marked (greater than 60%) inhibition of uptake of 125I-ovine prolactin (125I-oPRL) into Golgi light and intermediate fractions but no inhibition of uptake into Golgi heavy and plasmalemma elements. At times from 2 to 45 min postinjection, 125I-oPRL was extracted from Golgi elements and found to be largely intact as judged by rebinding to receptors. The inhibitory effect of colchicine was seen at doses ranging from 0.25 mumol to 25 mumol/100 g body wt. Vincristine also inhibited 125I-oPRL uptake into the Golgi light and intermediate fractions but lumicolchicine had no inhibitory effect. There was a smaller effect of colchicine both at early (1 h) and later (3 h) times on the extent and pattern of 125I-insulin uptake. Colchicine treatment did not produce a significant change in lactogen receptor levels in the Golgi fractions. These results demonstrate that colchicine treatment inhibited the transfer of prolactin into Golgi vesicular elements. The much smaller effect on insulin uptake suggests that there may be differences in the manner in which the two hormones are handled in the course of internalization.

Binding and internalization of 12Sl-ovine prolactin into hepatocytes of female rats was visualized by the in vivo radioautographic method (Bergeron, J. J. M., G. Levine, R. Sikstrom, D. O'Shaughnessey, B. Kopriwa, N. J. Nadler, and B. I. Posner, 1977, Prec. NatL Acad. Sci. USA, 745:051-5055). Receptor-mediated internalization of label was observed into lipoprotein-filled vesicles in the Golgi/bile canalicular region of the hepatocyte. Colchicine treatment had no effect on the internalization of label into the lipoprotein-filled vesicles. However, the location of the radio-labeled lipoprotein-filled vesicles was altered from the Golgi/bile canalicular region to subsinusoidal. Radioactive content of hepatocytes decreased as a function of time after injection of 12~l-prolactin; however, colchicine treatment markedly retarded this loss of label. Subcellular fractionation experiments indicated that colchicine treatment led to decreased levels of ~2~l-protactin accumulation in microsomes but augmented the accumulation of label in the L fraction.It is concluded that in normal female rats prolactin is internalized into lipoprotein-filled vesicles in the Golgi region before degradation of the hormone. Colchicine treatment accumulates labeled lipoprotein-containing vesicles in a subsinusoidal region and retards hormone catabolism. The labeled vesicles observed after colchicine treatment may correspond to the unique vesicles previously observed in the L fraction and found to be enriched in prolactin receptors