Detection and isolation of inactive, large molecular weight renin in human kidney and plasma.

Abstract

Estimation of apparent molecular weight (raw) of inactive renin by gel filtration of human plasma was found to be inaccurate when "acid activation" or "cryoactivation" was used for detection; recoveries were only 5% to 20%. Trypsln activation produced greater recoveries, but the apparent elution volume of inactive renin varied with the concentration of trypsln used; the presence of trypsln inhibitors increased trypsin requirements to 100 to 200 Mg/ml in the 60,000 dalton region, while low protein concentration in the 50,000 dalton region resulted in destruction of renin by as little as 10 Mg/ml trypsin. A composite trypsin-activated inactive renin peak corresponded to a raw of 56,000 ± 1500 daltons (104% to 120% recovery), while active plasma renin was 48,000 ± 2000 daltons. When this prorenln-like substance was isolated by affinity chromatography, it was found to be completely inactive. It was also nearly free of trypsin inhibitors, so that a single trypsin concentration correctly identified and confirmed the elution characteristics of inactive renin peak following gel filtration. The apparent mw of trypsin-activated inactive renin was slightly lower (52,500 daltons) than that of inactive renin.Human renal cortex was also found to contain a trypsin-actlvable form of renin. Like plasma inactive renin, it could be isolated by chromatography on Cibacron blue-agarose (Affi-Gel blue). It was found to be completely inactive following passage over a pepstatin affinity column. This inactive renal renin, as well as a similar substance in perfusate of normal human kidney, had a mw of 49,500 ± 1000, while active renal renin was 39,500 ± 500. Trypsin-activated inactive renal renin had a mw of 46,500 ± 500; its pH optimum was identical with that of active renal renin, and it no longer bound to Cibacron blue-agarose. We conclude that both human plasma and kidney contain an inactive, prorenin-like substance that can be detected reliably by trypsin activation. There appear to be slight differences in the apparent mw of plasma renins and kidney renin, but the similarity of other characteristics suggests that the inactive, prorenin-llke substances in renal cortex, renal perfusate, and plasma may be one and the same substance. (Hypertension 3 (suppl I): I-30-I-40, 1981) KEY WORDS • renin • prorenin • inactive renin • kidney • plasma affinity chromatography • molecular weight A N inactive form of renin (EC 3.4.99.19) inhuman plasma develops renin-like activity following limited proteolysis, including the processes of acid activation or cryoactivation. 13 As recently reviewed,' there has been considerable disagreement on the apparent molecular weight of inactive renin as determined by gel filtration of plasma, with values ranging from 43,000 to 63,000. 4 -10 These discrepancies may be related in part to the use of acid activation to detect inactive renin in chromatographic fractions, since the activation of inactive renin that occurs at pH 3 is reversible at a higher pH 11 ' 12 and the subsequent activation that occurs at alkaline ...

Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

“…These similarities have emerged despite the use of only partially purified preparations of plasma and renal inactive renin, which could contain variable quantities of enzymes and other proteins that might affect activation and characterization. These findings are consistent with the postulate that plasma inactive renin arises from the kidney and are supported (a) by the present observations demonstrating a gradient of inactive renin in IVC and renal vein blood and (b) by the report of Atlas et al (8) who found inactive renin in the perfusate of an isolated human kidney.…”

“…The use of different activation techniques may have resolved some of the molecular weight discrepancies previously reported between plasma and renal inactive renin (8,9). Several methods used in this study also emphasize the heterogeneity of human inactive renin.…”

“…The peaks in plasma correlated to those in kidney, 56,000±1,500 and 49,200±1,000. In contrast, Atlas et al (8) and Chang et al (9) described a single peak of plasma and renal inactive renin of 56,000-and 50,000-mol wt, respectively. Both groups used trypsin treatment to define inactive renin, and we have confirmed these estimates using trypsin.…”

“…Recent understanding of the properties and stability of inactive renin has lead to improved conditions of extraction of inactive renin from tissue sources. As a result, Atlas et al (8), Chang et al (9), and our laboratory (10) have reported the isolation of a completely inactive form of renin from human renal cortex. The purpose of this study was (a) to provide a detailed analysis of the quantitative relationship between active and inactive renin within the kidney and plasma; (b) to compare activation characteristics and biochemical properties of renal and plasma inactive renin; and (c) to determine whether renal inactive renin is released into the plasma.…”

Characterization of inactive renin ("prorenin") from renin-secreting tumors of nonrenal origin. Similarity to inactive renin from kidney and normal plasma.

“…Inactive renin can be activated in vitro by acidification to pH 3.3 or by treatment with certain proteolytic enzymes (1)(2)(3)(4)(5)(6)(7)(8). This substance has several chromatographic properties that clearly distinguish it from the active enzyme (8)(9)(10)(11)(12)(13) and it also has a slightly greater molecular weight by gel filtration (8,(12)(13)(14).…”