Isolation of luminal and antiluminal membranes from dog kidney cortex

Kinsella, James L.; Holohan, P D; Pessah, Ness I.; Ross, Charles R.

doi:10.1016/0005-2736(79)90191-3

Cited by 94 publications

(20 citation statements)

References 30 publications

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“…The cortex from both kidneys was homogenized at 4°C in Tris buffer (pH 7.0) containing 8% (wt/vol) sucrose in a glass-homogenizer with 10 strokes of a tight-fitting pestle. Basolateral (BL)' and luminal (L) membranes were prepared from this homogenate by the sequence of differential and gradient centrifugation combined with ionic precipitation described by Kinsella et al (19), with minor modifications. After centrifugation at 1,000 g (10 min) the pellet was rehomogenized and centrifuged for a second time (1,000 g; 10 min) (model RC2-B, DuPont InstrumentsSorvall Biomedical Div., DuPont Co., Newton, CT.).…”

Section: Methodsmentioning

confidence: 99%

“…Supernates were combined and spun at 9,500 g (10 min), and the resulting supernate and lighter pellet portion were centrifuged at 47,000 g for 20 min (model L ultracentrifuge, Beckman Instruments, Inc., Spinco Div., Palo Alto, CA.). The lighter pellet portions obtained after this step were resuspended in Tris Hepes Mannitol (THM) buffer (19) containing 2 mM CaCl2 and 1 mM each MgCl2 and MnCl2 and incubated on ice for 60 min. After ionic precipitation supernates contained the luminal membranes and the pellet consisted of crude basolateral fragments.…”

Section: Methodsmentioning

confidence: 99%

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Insulin Binding and Degradation by Luminal and Basolateral Tubular Membranes from Rabbit Kidney

Talor¹,

Emmanouel²,

Katz³

1982

J. Clin. Invest.

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A B S T R A C T Insulin influences certain metabolic and transport renal functions and is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to these events remains controversial. We studied 125I-insulin degradation [TCA and immunoprecipitation (IP) methods] and the specific binding of the hormone by purified luminal (L) and basolateral (BL) tubular membranes. These were prepared from rabbit kidney cortical homogenates by differential and gradient centrifugation and ionic precipitation steps in sequence, which resulted in enrichment vs. homogenate of marker enzymes' activities (sodium-potassium-activated adenosine triphosphatase for BL and maltase for L) of 8-and 12-fold, respectively. Both fractions degraded insulin avidly and bound the hormone specifically without saturation even at pharmacologic concentrations (10 lM). At physiologic insulin concentrations (0.157 nM) BL membranes degraded substantial amounts of insulin (44.2±2.6 and 40.7±2.2 pg/mg protein per min by the TCA and IP methods, respectively), even though at lesser rates (P <0.001) than the luminal fraction (67.2±2.3 and 75±6.2 pg/mg protein per min, respectively); the rate of insulin catabolism by BL membranes was significantly higher (P < 0.001) than that which could be attributed to their contamination by luminal components [12.2±1.9 pg/mg per min (TCA method), or 13.7±1.9 pg/mg per min (IP method)]. Competition experiments suggested that insulin-degrading activity in both fractions includes both specific and nonspecific components. In contrast to degradation, insulin binding by both membranes was highly specific for native insulin and was severalfold higher in BL than L membranes [17.5±1.3 vs. 4.5±0.4 fmol/mg protein (P < 0.001) at physiologic insulin concentrations]. Despite the marked difference in the binding capacity for insulin by the two membranes,

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Insulin Binding and Degradation by Luminal and Basolateral Tubular Membranes from Rabbit Kidney

Talor¹,

Emmanouel²,

Katz³

1982

J. Clin. Invest.

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“…In preliminary binding experiments, simultaneous preparation of luminal (BBM) and antiluminal (basolateral) membranes from pig kidney proximal tubule was performed as described by Kinsella et al (1979) Vannier et al (1976) and modified by Lin et al (1981). Briefly, after perfusion, cortex was removed from pig kidneys and placed in 10 mM mannitol, 2 mM Tris-HCl buffer pH 7.1.…”

Section: Preparation Of Renal Membranesmentioning

confidence: 99%

Characterization of adenosine receptors in brush‐border membranes from pig kidney

Blanco

Canela

Mallol

et al. 1992

British J Pharmacology

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“…This dissection was done with the kidneys on ice in a cold room. The method of membrane isolation was that of Kinsella et al [9] and Youmans et al [4] with modifications as previously described [6]. The medullas and cortices were placed in ice-cold 8% sucrose (pH 7.0), homogenized by a polytron for 10-30 s and then subjected to 10 strokes o f a motor-driven Potter-Elvehjem homogenizer.…”

mentioning

confidence: 99%

“…This aliquot was subsequently used to determine homogenate marker enzyme activities. The rest of the homogenate was subjected to extensive differential centrifugation followed by discontinuous sucrose gradient centrifugation as described by Kinsella [9]. The buffer used contained 100 mM mannitol and 25 mM Hepes with the pH brought to 7.0 with Tris base.…”

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confidence: 99%

Plasma membrane proton ATPase from human kidney

Sallman

Lubansky

Talor³

et al. 1986

European Journal of Biochemistry

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Distal urinary acidification is thought to be mediated by a proton ATPase (H+‐ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+‐ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+‐ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+‐ATPase) and in the absence of Ca (to inhibit Ca2+‐ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N‐ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4′‐diisothiocyano‐2,2′‐disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+‐ATPase, respectively. Dicyclohexylcarbodiimide and N‐ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward‐directed K gradient, there was increased quenching of acridine orange, indicating that the H+‐ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+‐ATPase, which is similar to the H+‐ATPase described in other species, and we postulate that this H+‐ATPase may be involved in urinary acidification.

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Isolation of luminal and antiluminal membranes from dog kidney cortex

Cited by 94 publications

References 30 publications

Insulin Binding and Degradation by Luminal and Basolateral Tubular Membranes from Rabbit Kidney

Insulin Binding and Degradation by Luminal and Basolateral Tubular Membranes from Rabbit Kidney

Characterization of adenosine receptors in brush‐border membranes from pig kidney

Plasma membrane proton ATPase from human kidney

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