A homologous biological probe for parathyroid hormone in human serum.

A method of measuring the biological activity of parathyroid hormone (PTH) in human serum that depends on the activation of its natural target enzyme, human renal cortical adenylate cyclase, is described. Optimal sensitivity ranging in different assays from 14 to 20 pg 1-34 hPTH/ml was achieved in the presence of the GTP-analogue GppNHp (10 mumol/L), 5 mmol/L MgCl2 and 1.25 mmol/L EGTA. Basal and stimulated cAMP production was reproducible within assays (c.v. below 7%, S.E.M., n = 3) and between assays (c.v. 5 to 14%, S.E.M., n = 4). The recovery of 1-34 hPTH added to individual test sera averaged 94%. The specificity of the method was established as follows: 1.) Other tested hormones, at 100 ng/ml, were ineffective; 2.) In the majority of peripheral sera from patients with hyperfunctioning parathyroid glands elevated bio-activity was detected; 3.) The circulating bio-activity fell rapidly after removal of parathyroid adenomata; 4.) Treatment with antisera for hPTH reduced the bio-activity; 5.) A PTH-antagonist inhibited the bio-activity.

Characterisation of the binding sites of anti-parathyroid hormone antisera using synthetic parathyroid hormone peptides.

Four antisera raised against partly purified PTH preparations all showed a wide range of specificities when reacting with radioiodinated PTH peptides representing several different portions of the intact hormone sequence. In contrast, antisera raised against individual peptides were only able to cross-react with other peptides that contained all or part of their amino acid sequence in common. Cross-reacting peptides were seen to contain one or more amino acid residues having high interspecies variability in common. We have explained the antigenicity and cross-reactivity of the peptides on the basis of these common highly variable amino acid sequences. We have concluded that the selection of hormonal material in radioimmunoassays for PTH should be made on the basis of the highly variable amino acid residue content. This will allow a narrowing of the assay specificities and permit detection of a desired region of the PTH hormone.

Homologous radioimmunoassay for human mid-regional parathyroid hormone.

A radioimmunoassay, selective for the clinically important mid region of human parathyroid hormone (hPTH), is reported. The synthetic 44-68 amino acid sequence (h44-68PTH) was used as both the standard and tracer material. This eliminated many of the undesirable characteristics associated with PTH assays the employ hormone of biological origin. These components allowed the detection of mid regional fragments present in both intact and fragmented hPTH, with a working range between 50 and 2500 pg/ml h44-68PTH. There was no interference from serum proteins and no significant cross reactivity to a range of N-terminal, C-terminal and other mid regional hPTH peptides. The assay proved to be rapid (total time 24 h) and was extremely reproducible, with an intraassay and interassay variation of 2.8% and 5.6% respectively (expressed as percentage SE in mean). The plasma concentration of normal subjects was established as 129 +/- 6 pg/ml (h44-68PTH) with a range of 50-300 pg/ml (n = 42). This assay, using fully synthetic hPTH peptides, was able to distinguish between euparathyroid and hyperparathyroid subjects, which suggests that the assay is of diagnostic value.

Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L. : I. Separation and characterisation.

The preparation is described of a cell-free system from developing cotyledons of Phaseolus vulgaris cv. Canadian Wonder which is capable of binding ethylene. The binding is saturable and the apparent dissociation constant for ethylene is 6.4·10(-10) M in solution. The binding site is associated with subcellular particles and treatment with Triton X-100 results in substantial solubilisation of the activity. The kinetics of association and dissociation of the ligand and the binding site are described. The system is heat labile and binding activity is diminished by treatment with some proteolytic enzymes.