REGION-SPECIFIC IMMUNOASSAYS FOR PARATHYROID HORMONE

1975 ◽  
Vol 66 (3) ◽  
pp. 307-318 ◽  
Author(s):  
P. M. BARLING ◽  
G. N. HENDY ◽  
M. C. EVANS ◽  
J. L. H. O'RIORDAN
Keyword(s):  
Parathyroid Hormone ◽  
Antigenic Site ◽  
Amino Terminal ◽  
Specific Radioimmunoassay ◽  
Lower Reactivity ◽  
Intact Molecule ◽  
Carboxy Terminal ◽  
Amino Terminal Fragment ◽  
Selection Of ◽  
Bovine Parathyroid Hormone

SUMMARY Immunoassays specific for limited regions of bovine parathyroid hormone were developed in four ways. With the heterogeneous antisera produced by immunizing with intact bovine parathyroid hormone (BPTH 1–84), the specificity of radioimmunoassays could be enhanced by presaturating either with an amino-terminal (BPTH 1–34) or carboxy-terminal (BPTH 53–84) fragment. Then, the antibodies which had not been neutralized reacted exclusively with the opposite end of the molecule, even using [125I]BPTH 1–84 as tracer. With some antisera, the appropriate fragment and intact hormone reacted identically. However, with other antisera, the fragment reacted less well than the intact hormone, possibly because these antisera contain antibodies reacting with the middle of the molecule. Using the labelled fragment ([125I]BPTH 1–34) as tracer, with heterogeneous antisera, radioimmunoassays specific for the amino-terminal region were obtained. With one antiserum, BPTH 1–34 reacted identically with the intact hormone, but with another antiserum, the fragment was more reactive than the intact molecule. A region-specific radioimmunoassay was also developed using antibodies produced by immunization with a fragment of the hormone. An antiserum raised against BPTH 1–34 had high affinity for the amino-terminal fragment, but reacted less well with the intact hormone. Immunoradiometric assays, specific for the amino- or carboxy-terminal regions, were developed by using immunoadsorbents consisting of a fragment (either BPTH 1–34 or BPTH 53–84) coupled to cellulose. These were used to fractionate 125I-labelled antibodies. With some of these selected antibodies, the appropriate fragment was of lower reactivity than the intact hormone. This may have been due to the presence of an incomplete antigenic site on the fragment, or to conformational differences between the fragment and the corresponding region of the intact hormone. With other selected antibodies the fragment and the intact molecule reacted identically. Careful selection of antisera and of technique is necessary to obtain an assay in which a fragment and the intact hormone behave identically.

THE IMMUNOCHEMICAL SPECIFICITY OF ANTISERA TO BOVINE PARATHYROID HORMONE: AN APPROACH TO REGION-SPECIFIC RADIOIMMUNOASSAY

1973 ◽  
Vol 56 (3) ◽  
pp. 493-501 ◽  
Author(s):  
T. M. MURRAY ◽  
H. T. KEUTMANN
Keyword(s):  
Parathyroid Hormone ◽  
Human Plasma ◽  
The Other ◽  
Amino Terminal ◽  
Specific Radioimmunoassay ◽  
Carboxyl Terminal ◽  
Bovine Parathyroid Hormone

SUMMARY Six different antisera to bovine parathyroid hormone were characterized immunochemically by comparing their reactivity towards amino-terminal (1–29) and carboxyl-terminal (53–84) fragments of the hormone molecule. Three of the antisera were primarily amino-terminally directed. One antiserum contained antibody populations to at least three discrete sites; two other antisera each contained two antibodies directed against each end region of the hormone. By saturating one of these antisera with one or the other of the end fragments of the hormone, specific immunoassays for the amino-terminal and carboxyl-terminal regions of the molecule were developed. Such techniques may be applied to improving the specificity of the radioimmunoassay for parathyroid hormone by eliminating interference from carboxyl-terminal hormonal fragments which circulate in human plasma.

scholarly journals Selective Uptake of the Synthetic Amino Terminal Fragment of Bovine Parathyroid Hormone by Isolated Perfused Bone

1978 ◽  
Vol 62 (2) ◽  
pp. 256-261 ◽  
Author(s):  
Kevin J. Martin ◽  
Jeffrey J. Freitag ◽  
Mary B. Conrades ◽  
Keith A. Hruska ◽  
Saulo Klahr ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Selective Uptake ◽  
Amino Terminal ◽  
Terminal Fragment ◽  
Amino Terminal Fragment ◽  
Bovine Parathyroid Hormone

Biologically active amino-terminal fragment of bovine parathyroid hormone prepared by dilute acid hydrolysis

Biochemistry ◽  
1972 ◽  
Vol 11 (10) ◽  
pp. 1973-1979 ◽  
Author(s):  
Henry T. Keutmann ◽  
Bess F. Dawson ◽  
Gerald D. Aurbach ◽  
John T. Potts
Keyword(s):  
Parathyroid Hormone ◽  
Acid Hydrolysis ◽  
Biologically Active ◽  
Dilute Acid ◽  
Dilute Acid Hydrolysis ◽  
Amino Terminal ◽  
Terminal Fragment ◽  
Amino Terminal Fragment ◽  
Bovine Parathyroid Hormone

Parathyroid hormone metabolism and its potential as a uremic toxin

1980 ◽  
Vol 239 (1) ◽  
pp. F1-F12 ◽  
Author(s):  
E. Slatopolsky ◽  
K. Martin ◽  
K. Hruska
Keyword(s):  
Renal Failure ◽  
Parathyroid Hormone ◽  
Chronic Renal Failure ◽  
Biological Effects ◽  
Uremic Toxin ◽  
Terminal Portion ◽  
Single Chain ◽  
Biochemical Alterations ◽  
Amino Terminal ◽  
Carboxy Terminal

Secondary hyperparathyroidism is a universal complication of chronic renal failure. It has been proposed that the markedly elevated levels of immunoreactive parathyroid hormone (i-PTH) in uremia may represent a “uremic toxin” responsible for many of the abnormalities of the uremic state. Plasma i-PTH consists of a mixture of intact hormone, a single-chain polypeptide of 84 amino acids, and smaller molecular weight hormonal fragments from both the carboxy- and amino-terminal portion of the PTH molecule. The hormonal fragments arise from metabolism of intact PTH by peripheral organs as well as from secretion of fragments from the parathyroid glands. The structural requirements for the known biological actions of PTH reside in the amino-terminal portion of the PTH molecule. Carboxy-terminal fragments, biologically inactive at least in terms of adenylate cyclase activation, hypercalcemia, or phosphaturia, depend on the kidney for their removal from plasma, and thus accumulate in the circulation in chronic renal failure. It is unknown at the present time if other biological effects of these carboxy-terminal fragments may contribute to some of the biochemical alterations observed in uremia. The most significant consequence of increased PTH levels in uremia is the development of bone disease characterized by osteitis fibrosa. In addition, it would appear that PTH plays an important role in some of the abnormal electroencephalographic patterns observed in uremia. This may be due to a potential role of PTH in increasing calcium content of brain. Parathyroid hormone also has been implicated as a pathogenetic factor in many other alterations present in uremia, i.e., peripheral neuropathy, carbohydrate intolerance, hyperlipidemia, and other alterations. Unfortunately, outstanding clinical research is lacking in this field and conclusive experimental data are practically nonexistent. Further studies are necessary if one is to accept the concept of PTH being a significant “uremic toxin.”

EFFECT OF GUANYL NUCLEOTIDES ON PARATHYROID HORMONE-RESPONSIVE ADENYLATE CYCLASE IN CHICK KIDNEY

1976 ◽  
Vol 69 (3) ◽  
pp. 401-412 ◽  
Author(s):  
N. H. HUNT ◽  
T. J. MARTIN ◽  
V. P. MICHELANGELI ◽  
J. A. EISMAN
Keyword(s):  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Enzyme Activation ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Amino Terminal ◽  
Nucleotide Analogue ◽  
Amino Terminal Fragment ◽  
Chick Kidney

SUMMARY Both guanosine 5′-triphosphate (GTP) and 5′-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase (EC 4.6.1.1) in chick kidney plasma membranes. Half-maximal stimulation occurred at 3·1 × 10−6 m for both agents. The maximum increases in adenylate cyclase activity produced by GTP and Gpp(NH)p were respectively 130 and 720% over basal activity. At the end of a 12 min incubation period GTP concentration was 85% of that originally added in the presence of an ATP-regenerating system but less than 20% in its absence. GTP and guanosine 5′-diphosphate inhibited the activation of adenylate cyclase by Gpp(NH)p, suggesting that they all acted at a common site. Gpp(NH)p facilitated the stimulation of adenylate cyclase activity by bovine parathyroid hormone (BPTH) and by the synthetic amino terminal fragment BPTH (1–34), decreasing the concentrations required for half-maximal enzyme activation by a factor of approximately eight in both cases. This property was not shared by the native nucleotide GTP. Gpp(NH)p rendered active (at certain concentrations) a synthetic parathyroid hormone peptide fragment, BPTH (2–34), which was incapable of activating adenylate cyclase in the absence of the nucleotide analogue. This suggested that the GTP analogue, in addition to a direct effect upon adenylate cyclase activity, was capable of influencing hormone interaction with the enzyme complex.

Immunologic Identification of the Cleavage Products from the Aα- and Bβ-Chains in the Early Stages of Plasmin Digestion of Fibrinogen

1986 ◽  
Vol 56 (01) ◽  
pp. 100-106 ◽  
Author(s):  
C Y Liu ◽  
Joan H Sobel ◽  
J I Weitz ◽  
Karen L Kaplan ◽  
H L Nossel
Keyword(s):  
Monoclonal Antibodies ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Small Peptides ◽  
Large Molecules ◽  
Amino Terminal ◽  
Specific Radioimmunoassay ◽  
Heterogenous Group ◽  
Reported Data ◽  
Carboxy Terminal

SummaryFragment X components (Mr 225,000 to 333,000) were distinguished on sodium dodecyl sulfate polyacrylamide gels. Western blotting with monoclonal antibodies to Aα-chain segments demonstrated that the Aα-chains of fibrinogen and the largest fragment X components (Mr 285,000-340,000) contained both Aα 259-276 and Aα 540-554. Fragment X components of Mr 270.000-285,000 contained Aα 259-276 but lacked Aα 540-554, whereas the smallest fragment X components (Mr 225.000-270,000) contained neither Aα 540-554 nor Aα 259-276. Studies of the small peptides generated during fragment X formation complemented the studies of the large molecules, by demonstrating peptides containing both Aα 259-276 and Aα 540-554 (Mr 41,600-41,800 and Mr 38,700-38,900), peptides containing Aα 540-554 but not Aα 259-276 (Mr 20,500-21,000 and Mr 17,300-17,500) and peptides containing only Aα 259-276 (Mr 23,600-24,000 and Mr 20,500-21,000). Cleavage of Bβ 1-42 from the amino terminal ends of the Bβ-chains, measured with a specific radioimmunoassay, was linear until 1.6 moles per mole of fibrinogen had been relased, and coincided with loss of the central and carboxy terminal Aα-chain regions, i. e. Aα 259-276 and Aα 540-554. Based on present and previously reported data, a model is proposed for the evolution of the heterogenous group of fragment X derivatives from fibrinogen with the simultaneous release of small peptides. Features of this model include1. asymmetric cleavage of the fibrinogen dimer and2. proteolyses of several different bonds occurring simultaneously but at distinct rates.

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