We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.

The sequence of the amino-terminal 37 residues of human parathyroid hormone has been established. The hormone used in these studies was isolated in highly purified form from parathyroid adenomata and was subjected to automated degradation in a Beckman sequencer. A high-sensitivity sequencing procedure employing 36S-labeled phenylisothiocyanate of high specific activity as the coupling agent was used. The sequence obtained differs from that of bovine parathyroid hormone in three of the first 37 positions, and from that of porcine parathyroid hormone in two positions. A single human-specific residue was found (asparagine 16). The sequence obtained differs at three positions (22,28, and 30) [(1973) Helv. Chim. Acta, 56, We have carefully reviewed our data, reported here in detail, on the sequence positions in dispute. We must conclude, on the basis of all available data, that the structure that we propose is the correct structure. The objective resolution of these discrepancies in structural analysis through further chemical and immunochemical studies is important, since synthesis of human parathyroid hormone, in which there is widespread interest for physiological and clinical studies, must be based on the correct sequence of the human hormone if the peptide is to be genuinely useful.Substantial advances have been made in recent years in our knowledge of parathyroid hormones, through studies of primary structure (1-3), structural requirements for biological activity (4, 5), biosynthesis (6-8), and metabolism (9)(10)(11)(12)(13)(14). Most of these studies, including the development and application of radioimmunoassays capable of measuring plasma parathyroid hormone levels in man, have depended directly or indirectly upon the use of the bovine and porcine hormones. Purified human parathyroid hormone (HPTH), on the other hand, has been available in microgram quantities, sufficient only for limited studies of its chemical and immonological properties (15).Recent improvements in extraction and isolation techniques, and the development of high-sensitivity methods for peptide sequence analysis have permitted us to determine the amino-acid sequence of the amino-terminal biologically active portion of HPTH (Fig. 1).After the submission for publication in abstract form of our findings for the N-terminal 31 residues of HPTH (24), the report of Brewer et al. (29) of their own independent structural studies on HPTH was published. Marked discrepancies between the two structures, which differ in three of the first 30 residues, have prompted us to reexamine our data for each cycle of the several degradations performed with the phenylisothiocyanate method.We now report in full the strategy and methods used in our sequence analysis as well as the quantitative aspects of the results and discuss the nature, implications, and possible approaches to resolution of the differences between the findings of Brewer et al. (29) and ourselves concerning the sequence of the amino-terminal portion of human parathyroid hormone. MATE...

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Sequence analysis suggests that KALRN, a Rho GDP/GTP exchange factor genetically linked to schizophrenia, could contain as many as nine tandem spectrin repeats (SRs). We expressed and purified fragments of Kalirin containing from one to five putative SRs in order to determine whether they formed nested structures that could endow Kalirin with the flexible rod-like properties characteristic of spectrin and dystrophin. Far UV circular dichroism studies indicated that Kalirin contains nine SRs. Based on thermal denaturation, sensitivity to chemical denaturants and the solubility of pairs of repeats, the nine SRs of Kalirin form nested structures. Modeling studies confirmed this conclusion and identified an exposed loop in SR5; consistent with the modeling, this loop was extremely labile to proteolytic cleavage. Analysis of a di-repeat fragment (SR4:5) encompassing the region of Kalirin known to interact with NOS2, DISC-1, PAM and Arf6 identified this as the least stable region. Analytical ultracentrifugation indicated that SR1:3, SR4:6 and SR7:9 were monomers and adopted an extended conformation. Gel filtration suggested that ΔKal7, a natural isoform which includes SR5:9, was monomeric and was not more extended than SR5:9. Similarly, the nine SRs of Kal7, which was also monomeric, were not more extended than SR5:9. The rigidity and flexibility of the nine SRs of Kal7, which separate its essential N-terminal Sec14p domain from its catalytic domain, play an essential role in its contribution to the formation and function of dendritic spines.

Using a method for continuous removal of cisternal cerebrospinal fluid (CSF) from freely moving cats, we delineated the circadian nature of the daily rhythm in CSF arginine vasopressin. The daily melatonin rhythm was also monitored in CSF as another marker of circadian function. Under diurnal lighting conditions, both hormones exhibited prominent daily rhythms; the CSF vasopressin rhythm was characterized by high daytime values, whereas the CSF melatonin rhythm was characterized by high nighttime levels. In contrast, drinking behavior exhibited a 24-h component in only one of four animals studied. Daily CSF rhythms of vasopressin and melatonin persisted for over 78 days of study in constant light. The vasopressin rhythm clearly free-ran in this environment, manifesting cycle lengths of slightly greater than 24 h. The daily melatonin pattern split into several components with increasing time in constant light. An acute 8-h phase delay in the daily light-dark cycle resulted in corresponding but gradual phase shifts in both rhythms. These results indicate that both the vasopressin and melatonin rhythms in cat CSF are endogenously generated and are entrained by the daily light-dark cycle.

A rabbit antiserum to somatostatin was used to develop radioimmunoassay methods for measuring somatostatin in monkey cerebrospinal fluid (CSF). By gel permeation chromatography, at least five molecular weight forms of immunoreactive somatostatin (IRS) were identified in monkey CSF; two of these species co-migrated with either synthetic somatostatin-14 or somatostatin-28. The 24-hr profile of CSF somatostatin immunoreactivity was obtained from five monkeys during diurnal lighting, constant light, and constant darkness. During diurnal lighting, all five monkeys had a clear ultradian component in CSF IRS of 4 to 5 hr duration; this pattern was not affected significantly by constant light or dark. In addition, there of the five monkeys exposed to diurnal lighting showed a diurnal rhythm in CSF IRS, with higher hormone levels during darkness. In some animals, this diurnal rhythm also could be demonstrated during constant light or dark.

During the isolation of human parathyroid hormone there is an extensive loss of immuno-assayble hormone over the successive extraction steps, due in part to the presence of fragments that are soluble in 4% trichloroacetic acid. These fragments are derived from both the amino- and carboxyl-terminal regions of the hormone. The hormonal fractions precipitated with trichloroacetic acid were further purified by gel filtration and ion-exchange chromatography. At the final ion-exchange purification step, some preparations of the hormone eluted in multiple fractions. When the various components were characterized separately by immunoassay, amino acid composition, enzymic cleavage and partial sequence analysis, they were found to be closely comparable, although the most acidic fraction contained a blocked terminal amino group. Extraction of a number of batches of tissue permitted revision of the amino acid composition of human parathyroid hormone. Biosynthetic studies with labelled amino acids confirmed the absence of tyrosine and the presence of phenylalanine and threonine and localized these residues to definite regions of the molecule.

Vitamin D-deficient rats subjected to thyroparathyroidectomy (TPTX) were used to evaluate in vivo the biological properties of native bovine parathyroid hormone (bPTH) and chemically synthesized fragments and analogues of the hormone on several parameters of hormone action: calcium and phosphorus fluxes, generation of cyclic adenosine 3',5'-monophosphate (cAMP), and the metabolism of 25-hydroxyvitamin D3 [25(OH)D3]. Vitamin D-deficient rats, after TPTX or sham operation, were intravenously infused with a nutrient containing 7.5 mM CaCl2 for 30 h. During the last 7 h, PTH or one of its analogues was infused intravenously at rates between 0.04 and 20 nmol/h. One hour after the start of the peptide infusion, tritiated 25(OH)D3 was injected. Urine was collected hourly for phosphate and cAMP determinations and, at the end of the experiment, blood was obtained to determine the relative accumulation of tritiated 1,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3). Infusion of bPTH-(1--84), bPTH-(1--34), human (h)PTH-(1--34), or [Nle8, Nle18, Tyr34]bPTH-(1--34) amide was accompanied by a comparable dose-dependent decrease in plasma phosphate and a dose-dependent increase in plasma calcium and [3H]-1,25(OH)2D3, and urinary excretion of phosphate and cAMP. An evaluation of [Nle8, Nle18, Tyr34]bPTH-(3--34) amide, a potent inhibitor of PTH action in vitro in the renal adenylate cyclase assay, revealed that the analogue possessed weak agonist properties in vivo. The analogue increased excretion of both cAMP and phosphate in the urine, decreased plasma phosphate levels, and increased the accumulation of [3H]-1,25(OH)2D3 in the plasma. This multiparameter model system should aid in the elucidation of the in vivo biological effects of PTH and its analogues.