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.
Parathyroid hormone (PTH) is rapidly metabolized, mainly by liver and kidney, to smaller fragments that are believed to be biologically inactive. The significance of this peripheral metabolism in the overall actions of PTH is unclear. Generation of circulating biologically active amino-terminal PTH fragments during metabolism in vivo has been suggested by certain observations in vitro, and what are believed to be amino-terminal fragments may be detectable in blood under pathological circumstances in vivo (such as renal failure and coexistent hyperparathyroidism) when highly sensitive assays are employed. We recently reported, however, that administration to normal rats of [35S]bovine PTH ([35S]bPTH) directly labelled at amino-terminal methionines, followed by high-resolution chromatographic analysis of extracted [35S]peptides, does not result in appearance of radioactive amino-terminal fragments in blood, even when the tracer is continuously infused to near-physiological plasma concentrations. We have now employed these techniques to address a second question regarding hormonal metabolism: is hormonal metabolism modified during metabolic perturbations such as changing calcium availability or altered levels of calciotrophic hormones? Metabolism of [35S-Met]bPTH (900 Ci/mmol), either alone or together with [3H-Pro]bPTH, however, did not lead to alterations in the rate of hormonal clearance nor to detectable circulating amino-terminal fragments, either in calcium-deprived or thyroparathyroidectomized rats or when animals were first rendered intoxicated with vitamin D or maintained on a high calcium intake. Likewise, tissue localization and specific cleavage patterns of intact hormone in liver or kidney were all unaltered by these various manoeuvres.(ABSTRACT TRUNCATED AT 250 WORDS)
Treatment of cystinuria with D-penicille, introduced by Crawhall, Scowen, and Watts (1963), has dramatically reduced morbidity due to cystine urolithiasis in selected patients, both by preventing new stone formation (Crawhall, Scowen, and Watts, 1964; Bartter, Lotz, Thier, Rosenberg, and Potts, 1965;MacDonald and Fellers, 1966) and by dissolving stones lodged in the renal pelvis (Lotz and Bartter, 1965;McDonald and Henneman, 1965). These effects were apparently based on formation of the more soluble mixed disulphide, penicillaminecysteine, in place of cystine. However, the administration of D-penicillamine involves a definite risk of complications, such as reactions resembling serum sickness, skin rash, nephrotic syndrome, agranulocytosis, impairment of taste sensation, and iron depletion. In addition, D-penicillamilne inactivates pyridoxal-5-phosphate and gives rise to biochemical changes typical of pyridoxine deficiency in experimental animals (Asatoor, 1964) and in man (Jaffe, Altman, and Merryman, 1964). It also affects collagen metabolism, producing inhibition of wound-healing in normal rats (Nimni and Bavetta, 1965) and in patients after long-term treatment for Wilson's disease or cystinuria (Scheinberg, 1964;Harris and Sjoerdsma, 1966).All three of the reactive sites of the D-penicillamine molecule -the a-amino group, the sulphydryl group, and the terminal carboxyl group-are required for maximal metal-chelating activity (Aposhian, 1961); interference with pyridoxal phosphate involves at least two, the a-amino and the sulphydryl (du Vigneaud, Kuchinskas, and Horvath, 1957 Henry, Sobel, and Chiamori (1958), respectively. In six patients urinary excretion of kynurenine during 24 hours after a 2-g. oral load of L-tryptophan was determined by the method of Tompsett (1959). N-acetyl-penicillamine, 2 to 4 g./day by mouth in equally divided six-hourly doses, was administered for a period of five days to five weeks. Haematological, liverfunction, and tryptophan-loading tests were repeated at weekly intervals; serum copper, ceruloplasmin, and inulin and P.A.H.clearances were determined on one or more occasions; and the 24-hour urinary output of free cystine, N-acetylpenicillamine-cysteine mixed disulphide and cysteine-homocysteine mixed disulphide was measured daily or at intervals. Similar studies were also carried out in eight patients during a course of D-penicillamine 2 to 4 g./day.The procedures for collecting 24-hour urine specimens, for determining renal clearances, and for measuring cystine and mixed disulphides in urine and plasma were as described elsewhere (Stokes, Potts, Lotz, and Bartter, 1966a, 1968), except for a modification of the automatic amino-acid analyser system necessary for the determination of homocysteine-cysteine; elution at 550 C. with 0.2 M sodium citrate pH 3.5 buffer (68 ml./hour) was followed after 30 minutes by 0.2 M citrate pH 4.1 buffer containing 2.3% benzyl alcohol and 5% propanol. ResultsCharacteristics of N-acetyl-D-penicillamine and N-acetyl-D-penicillamine-cysteine N-ace...
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