Isolation of renin granules from the dog kidney cortex.

Morimoto, Shiro; Yamamoto, Kenjiro; Ueda, Juro

doi:10.1152/jappl.1972.33.3.306

Cited by 33 publications

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“…The renin was thereby enriched about ninefold over the original homogenate at its peak activity in the part of the density gradient corresponding to 1.5-1.6 M sucrose. Similar results have also been described not only for rat* but for dog 23 and rabbit"-" renal renin. Whereas on gradient centrifugation of the heavy mitochondrial fraction a broad peak of renin activity was observed, during density gradient centrifugation of the total postnuclear supernatant in the B XIV zonal rotor, the particulate renin was clearly separated from the other marker enzymes ( fig.…”

Section: Effect Of N-ethylmaleimlde On Gel Filtration Of Renin From Ssupporting

confidence: 86%

Subcellular distribution and storage form of rat renal renin.

Sagnella¹,

Price²,

Peart³

1980

Hypertension

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SUMMARYThe subcellular distribution and nature of rat renal renin has been Investigated by means of analytical subcellular fractionation and gel filtration on Sephadex G-100. During differential centrifugation, renin activity was recovered mainly in soluble and heary mltochondrial fractions. On sucrose gradient centrifugation in either a conventional or in a B XIV zonal rotor, renin activity equilibrated at 1.54 M sucrose and was partially resolved from marker enzymes for mitochondria (succinate debydrogenase), lysosomes (acid phosphatase), plasma membranes (alkaline phosphatase), and peroxisomes (catalase). On gel filtration of the soluble or extracts of the renin-granular fractions on Sepbadex G-100, renin activity eluted as a single peak with an apparent molecular weight (MW) of 42,000; no change In activity was found when these fractions were acidified to pH 3.0. When kidney homogenates were prepared in the presence of the proteolytic inhibitor Nethylmaleimide (NEM, 10 mM), whereas the renin from the granular fractions displayed a MW of 44,000, that from the soluble fraction was apparently higher (69,000). Addition of NEM (10 mM) to the soluble fraction previously shown to contain only the low MW form of renin also resulted in an apparently high MW form of renin.These results indicate that rat renal renin is associated with a mechanically fragile, distinct type of subcellular organelle. Renin within this structure is of the low MW form and is not acid activatable. 3 "* have been contradictory as to the precise subcellular distribution of the enzyme. Also, limited information is available on the nature of the storage form of rat renin. It has been reported, however, that rat renin is stored as an inactive form that can be activated by acidification to pH 3.3,' yet others 7 '' have been unable to demonstrate acid activatable forms of rat renin.Recently, Inagami et al. 9 found that, in the presence of the proteolytic inhibitor N-ethylmaleimide, only a high molecular weight (HMW) form of renin (60,000) was extracted from the rat kidney; consequently they concluded that this form of renin represented a precursor of the low molecular weight (LMW) form of the enzyme. However, since these studies were carried out with crude soluble cortical extracts, the relationship between this HMW form of renin and the granular renin remained undetermined.Therefore, it was thought of importance to investigate further the subcellular distribution of renin and in particular to determine the effect of acidification and N-ethylmaleimide on the activity and molecular size of renin not only from soluble but also from granular fractions. 595 Materials and Methods Preparation of Cortical HomogenatesMale Wistar rats (180-200 g) that had free access to water and standard laboratory food were killed by cervical dislocation. The kidneys were removed, stripped of the capsule and bisected. The cortices were

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Section: Effect Of N-ethylmaleimlde On Gel Filtration Of Renin From Ssupporting

confidence: 86%

Subcellular distribution and storage form of rat renal renin.

Sagnella¹,

Price²,

Peart³

1980

Hypertension

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“…This gradient was selected because dog renin granules are mainly recovered in the interphase of 1.5 and 1.6 M sucrose. 17 After centrifugation at 60,000 g for 90 minutes, the gradient had two visible bands and some sediment. The gradient was separated into three fractions, F0 (volume 25.2 ± 0.3 ml), Fl (volume 12.6 ± 0.2 ml), and F2 (volume 18.9 ± 0.2 ml) from the top of tube, by using a ISCO Model 185 fractionator (Instrumentation Specialties Company, Nebraska).…”

Section: Blood Sampling and Preparation Of Subcellular Fractionation mentioning

confidence: 99%

Effects of sodium depletion on inactive and active renin from dog kidney and plasma.

Kawamura¹,

Akabane²,

Ito³

et al. 1984

Hypertension

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SUMMARY The relationship of active renin and inactive renin (trypsin-activated angiotensin-I-forming enzyme) to sodium depletion was examined in renal and peripheral plasma and at the subcellular level in the kidneys of dogs. Subcellular fractionation was carried out by discontinuous sucrose density (1.5 and 1.6 M) centrifugation. Sodium depletion selectively caused a six-to sevenfold increase in the renal content of inactive and active renins in the original homogenate, while the subcellular distribution patterns of these enzymes were little changed. Of the total granule fractions of 1.5 M sucrose (Fl), 1.6 M sucrose (F2), and sediment (F3), approximately 80% of inactive renin was recovered in Fl, which was rich in microsomes, while about 50% of active renin was in F2. The ratio of inactive to active renin was 0.02 in Fl and 0.003 to 0.004 in F2. Sodium depletion also caused a 20-fold increase in active renin and a twofold increase in inactive renin in peripheral plasma. The renal venous-arterial concentration difference of inactive renin was statistically significant in lowsodium dogs, although it was not significant in controls. The ratio of inactive to active renin was 0.2 to 0.4 in plasma from low-sodium dogs, while it was 1.5 to 3 in plasma from control dogs. These results suggest that plasma inactive renin originates, at least in part, in the kidney. T is now widely accepted that human plasma contains an inactive form of renin, which can be activated with trypsin. 1 -2 However, little is known of the physiological mechanisms controlling the activation of inactive renin. Recently, inactive renin was detected in human kidney extracts 3 " 5 and also in the renin granules.6 Plasma inactive renin is thought to originate, at least to some extent, in the kidney, although an extrarenal origin has also been suggested. "9 In laboratory experiments, inactive renin was detected in dog 10 and rat"-l2 plasma and dog 10 and hog 13 kidneys. It is well known that plasma active renin usually reflects the active renin content of the kidney. In particular, sodium intake is a major determinant in con- trolling the active renin in plasma and kidney. 1415However, only limited information exists on the responses of inactive renin to sodium depletion in both the plasma and kidney. As renin is known to be synthesized in a precursor form in the microsomal fraction, 16 the inactive renin content of the kidney should aiso be examined at subcellular levels. Therefore, we have evaluated the effect of sodium restriction on renin concentration of renal and peripheral plasma and on subcellular distribution of kidney renin. Material and Methods Experimental ProtocolSixteen male mongrel dogs weighing 18 to 20 kg were kept in metabolic-balance cages and given tap water ad libitum. For over 2 weeks before the study, the animals were fed a standard laboratory dog chow (Japan Clea Company, Osaka, Japan) containing 57.5 mEq sodium and 88 mEq potassium per daily intake. Each daily urine collection was analyzed for sodium and potassium contents. A...

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“…Therefore, it seems that the higher percent of active renin in extracts from frozen kidney is not due to activation of inactive renin by freezing and thawing. Freezing releases enzymes from intracellular organelles such as lysosomes and zymogen granules (8,9). Possibly, extracts of fresh kidney contain some renin that is membrane bound and inaccessible to substrate.…”

Section: Extraction Of Renin Frommentioning

confidence: 99%

A renin inhibitor from rabbit kidney: conversion of a large inactive renin to a smaller active enzyme.

Leckie¹,

McConnell²

1975

Circulation Research

100

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Renin in extracts of frozen rabbit kidney exists in two forms: active (molecular weight about 37,000) and inactive (molecular weight about 55,000) renin. The inactive form becomes active after exposure to pH 2.5 at 4°C. If extracts are chromatographed on DEAE cellulose, the inactive renin dissociates into active renin plus a renin inhibitor (molecular weight about 13,000). The inhibitor recombines with active renin if the two are incubated together at 37°C. The inhibitor is destroyed by acid treatment at pH 2.5 at 4°C. We conclude that the activation of inactive renin is due to destruction of the inhibitor by acid. The inactive material may be a renin proenzyme or a storage form of active renin combined with inhibitor. KEY WORDS renin pressor responseproenzyme renin activation molecular weight of renin membrane-bound renin• Extracts of rabbit kidney contain an active and an inactive form of the enzyme renin (1). Inactive rabbit renin has a molecular weight of around 55,000, and active renin has a molecular weight of around 37,000. When a solution of inactive rabbit renin is acidified to pH 2.5, its pressor activity increases and its molecular weight becomes similar to that of active rabbit renin. A slow-acting renin is also present in extracts of pig kidney (2). It is activated by acid and dissociates into active renin plus a "renin-binding protein," when it is chromatographed on DEAE cellulose. The inactive renin from the rabbit kidney may be similar to the slow-acting renin from the pig kidney.The present experiments were carried out in an attempt to characterize the inactive renin from rabbit kidney and to study its activation. MethodsKidney Extracts.-Kidneys were obtained from male or female New Zealand white rabbits. The rabbits were anesthetized with sodium pentobarbital, and their kidneys were excised and stored frozen. The kidney cortex was extracted as previously described (1). Extracts for DEAE cellulose chromatography were made in 0.005M sodium phosphate buffer, pH 7.0, using 3 ml buffer/g kidney cortex.Buffers.-Buffer solutions were boiled and made up to volume in boiled, cooled distilled water. Phosphatesaline buffer contained 0.05M NaHjPO 4 /Na 2 HPO 4 , 0.1M NaCl, and 2.0 g/liter of neomycin sulfate, pH 5.0-7.4. Glycine-HCl buffer contained 0.05M glycine/HCl and 0.1M NaCl, pH 1.5-4.0.From the MRC Blood Pressure Unit, Western Infirmary, Church Street, Glasgow Gil 6NT, Scotland.Received October 7, 1974. Accepted for publication January 13, 1975.Standard Renin.-Standard renin was prepared by a modification of the method of Lever et al. (3). Rabbit kidneys were minced in 0.9% saline and allowed to stand at 4°C for 48 hours. The slurry was centrifuged at 2,000 g for 30 minutes, the supernatant fluid was decanted, the precipitate was washed with half of the original volume of saline, and the washings were added to the supernatant fluid. All subsequent operations were carried out at 4°C. The extract was acidified to pH 2.5 and allowed to stand for 7-16 hours. The pH was adjusted to 7.4, the preci...

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Isolation of renin granules from the dog kidney cortex.

Cited by 33 publications

References 0 publications

Subcellular distribution and storage form of rat renal renin.

Subcellular distribution and storage form of rat renal renin.

Effects of sodium depletion on inactive and active renin from dog kidney and plasma.

A renin inhibitor from rabbit kidney: conversion of a large inactive renin to a smaller active enzyme.

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