Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell

1994 ◽  
Vol 72 (3-4) ◽  
pp. 157-162 ◽  
Author(s):  
Makoto Arao ◽  
Toru Yamaguchi ◽  
Toshitsugu Sugimoto ◽  
Masaaki Fukase ◽  
Kazuo Chihara
Keyword(s):  
Parathyroid Hormone ◽  
Molecular Mass ◽  
Hydrolytic Activity ◽  
High Molecular Mass ◽  
Phenylmethylsulfonyl Fluoride ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cell ◽  
Hydrolysis Of

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Sue, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from α-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250–300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1–84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.Key words: opossum kidney, parathyroid hormone, chymotrypsin, endopeptidase.

A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone

1987 ◽  
Vol 253 (2) ◽  
pp. E221-E227 ◽  
Author(s):  
J. A. Cole ◽  
S. L. Eber ◽  
R. E. Poelling ◽  
P. K. Thorne ◽  
L. R. Forte
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Rank Order ◽  
Phosphate Transport ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Camp Formation

Regulation of phosphate transport by parathyroid hormone (PTH) was investigated in continuous lines of kidney cells. Phosphate transport was reduced by PTH-(1-34) at physiological concentrations (EC50 5 X 10(-11) M), whereas much higher concentrations were required to stimulate cAMP formation (EC50 1 X 10(-8) M) in opossum kidney (OK) cells. The PTH analogue [Nle]PTH-(3-34) also inhibited phosphate transport but did not enhance cAMP formation. Instead, [Nle]PTH-(3-34) was a competitive antagonist of PTH-(1-34) at cyclase-coupled receptors. PTH-(7-34) had no effect on phosphate transport or cAMP formation. Phorbol esters or mezerein were potent inhibitors of phosphate transport but did not affect cAMP synthesis. Their potencies paralleled the rank-order potency of these agents as activators of protein kinase c in other systems. Maximally effective concentrations of PTH-(1-34) and mezerein did not produce additive inhibition of phosphate transport in OK cells. Phorbol esters stimulated phosphate transport in JTC-12 cells, but PTH-(1-34) had no effect. We concluded that PTH regulates OK cell phosphate transport by interacting with two classes of receptors, and transmembrane-signaling mechanisms. Physiological levels of PTH-(1-34) may regulate phosphate transport by activation of protein kinase c, whereas higher concentrations appear to activate adenylate cyclase.

Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis

1992 ◽  
Vol 127 (3) ◽  
pp. 267-270 ◽  
Author(s):  
Toru Yamaguchi ◽  
Makoto Arao ◽  
Masaaki Fukase
Keyword(s):  
Parathyroid Hormone ◽  
Biologically Active ◽  
Main Role ◽  
Kidney Cells ◽  
Binding Studies ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cell ◽  
Opossum Kidney Cells

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0× 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1-34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

Parathyroid Hormone Degradation by Chymotrypsin-Like Endopeptidase in the Opossum Kidney Cell*

Endocrinology ◽  
1988 ◽  
Vol 123 (6) ◽  
pp. 2812-2817 ◽  
Author(s):  
TORU YAMAGUCHI ◽  
MASAAKI FUKASE ◽  
MASASHI NISHIKAWA ◽  
TADAO FUJIMI ◽  
TAKUO FUJITA
Keyword(s):  
Parathyroid Hormone ◽  
Kidney Cell ◽  
Opossum Kidney ◽  
Opossum Kidney Cell
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