Shigehisa Hirose

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.

New fluorogenic substrates for renin

Abstract A simple and sensitive fluorometric assay was developed to test renin activity within several hours. Two new fluorogenic peptides, Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (octapeptide-MCA) and a succinyl derivative of the octapeptide-MCA were synthesized and used as a renin substrate. Renin cleaved the substrates at the Leu-Leu bond, releasing Leu-Val-Tyr-MCA. Three amino acids of this product were then successively split off by the auxiliary enzyme, leucine aminopeptidase, to liberate free 7-amino-4-methylcoumarin (AMC). The generation of the fluorescent 7-amino-4-methylcoumarin was proportional to renin concentrations up to 100 mGoldblatt U/tube. The optimal pH of renin reaction for both substrates was 6.5 to 7.0. As low as 5 mGoldblatt U of renin could be detected by this method. This method was applied to the assay of renin during its purification.

An improved method for determination of active and total renin concentration in human plasma using an excess of sheep substrate

A method for measurement of renin concentration (PRC) in human plasma has been developed and validated experimentally and theoretically. Like most of the previous methods, the present method is based on a radioimmunoassay of angiotensin I generated during incubation of plasma with an excess of renin substrate. In the present study we used, as the substrate, sheep angiotensinogen partially purified from anephric sheep plasma by ammonium sulfate fractionation and pepstatin-aminohexyl-agarose chromatography. The advantage of using sheep substrate is that it has an exceptionally high affinity for human renin. The partially purified substrate, which contained no detectable renin activity, improved the sensitivity, allowing quantification of renin in low-renin plasma. Possible interfering influences of plasma proteins on the radioimmunoassay were eliminated by the introduction of a simple deproteinization step. For determination of total renin concentration (TRC), inactive renin was activated by exposing plasma to low pH or trypsin. Normal values of PRC and TRC after careful selection of assay conditions were 2.9 +/- 0.4 and 33.9 +/- 6.1 ng angiotensin I X ml-1 X h-1, respectively. As an illustrative example of usefulness of the assay, PRC determination of a patient with a ectopic renin-secreting tumor is presented.

Demonstration of the formation of renin and renin-binding protein complex using high-performance liquid chromatography

A high-performance liquid chromatograph equipped with the newly developed gel, TSK G3000SW, was used to study the interaction between renin and renin-binding protein (RBP). Previously, the interaction could only be demonstrated after overnight gel chromatography in the presence of a non-physiological sulfhydryl reagent. However, this new high-speed gel chromatography provided a clear separation of renin and renin--RBP complex within 40 min. It also demonstrated that the renin--RBP complex was formed at 37 degrees C in the absence of sulfhydryl reagent. Theses results indicate that the binding protein may play an important role in blood pressure regulation.

Cell-Free Translation of Human Renin Mrna

The molecular weight of human renin precursor (preprorenin) was determined to be 43,000 by cell-free translation of its mRNA. Poly(A)+-containing RNA from human infarcted kidney was translated in a cell-free reticulocyte lysate system containing [35S] methionine. Immunoprecipitation with anti-renin antibody gave one major band with an apparent Mr = 43,000, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. The specific band, which represents the prepro-form of human renal renin, was very similar to mouse submandibular gland preprorenin in its electrophoretic mobility.

Properties of Renin-Binding Protein

Hog renal renin-binding protein could bind homologous and non-homologous renin but could not bind human renal renin. Renin-binding protein was only detected in the kidney and pituitary in which renin is found in relatively high concentration. The renal binding protein had no interaction with renal acid proteases nor with extrarenal renins obtained from pituitary and submaxillary glands, indicating that the binding is specific for renal renin. Subcellular localization of the binding protein was studied using rat kidney by differential and density gradient centrifugation. Most renin-binding protein was recovered in the cytosol fraction and was not associated with sedimentable subcellular organelles. A renin-secreting tumor (Juxtaglomerular cell tumor) in human kidney produced not only renin but also renin-binding protein in a very large quantity.

Structure of angiotensin II-binding site probed with diaminoalkanes

The nature of the hormone-binding site of detergent-solubilized angiotensin II receptor from the bovine adrenal cortex has been characterized using diaminoalkanes. Their affinity for the hormone-binding site showed a dramatic alteration depending on their chain length; at the hexamethylene stage, a maximum and competitive inhibition was observed. These results indicate that the angiotensin II receptors have a definite hydrophobic binding groove limbed with diagonally located two negative charges, and those hydrophobic and electrostatic forces may play an important role in the proper register between the receptor and hormone.