Enzymatic and transport characteristics of isolated snake renal brush-border membranes

S. Benyajati, William H Dantzler

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Abstract

Brush-border membranes (BBM) of proximal tubules were isolated from the kidney of the garter snake (Thamnophis sirtalis) by a procedure involving hypotonic lysis, Ca precipitation, and differential centrifugation. The isolated membranes were enriched 15-fold in brush-border enzyme activities (alkaline phosphatase, γ-glutamyl transpeptidase) compared with whole kidney homogenates and were substantially free of other contaminating membranes. The yield of the BBM preparation was 40%. The BBM vesicular transport of several organic solutes was characterized by a rapid filtration technique at 25°C. D-glucose, p-aminohippurate (PAH), and urate entered the same osmotically active space (2-3 μl/mg protein) and binding was minimal (<20% for PAH). An uptake overshoot for 3-O-methyl-D-glucose (20 μM) by reptilian BBM was observed only in the presence of an inwardly directed NaCl gradient and was abolished by 0.1 mM phlorizin. Reptilian BBM exhibited Na-gradient-stimulated uptake of PAH (90 μM) with an overshoot that was inhibited by other organic acids and by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). In contrast, urate uptake (30 μM) appeared to be Na independent and not appreciably affected by other organic anions or SITS. The presence of specific transport systems for organic solutes in the isolated membrane preparation distinctly characterizes the BBM of reptilian kidney.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
Volume255
Issue number1
StatePublished - 1988

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Snakes
Microvilli
Kidney
Membranes
p-Aminohippuric Acid
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
Colubridae
Uric Acid
3-O-Methylglucose
Phlorhizin
Acids
gamma-Glutamyltransferase
Centrifugation
Protein Binding
Anions
Alkaline Phosphatase
Glucose
Enzymes

ASJC Scopus subject areas

  • Physiology

Cite this

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abstract = "Brush-border membranes (BBM) of proximal tubules were isolated from the kidney of the garter snake (Thamnophis sirtalis) by a procedure involving hypotonic lysis, Ca precipitation, and differential centrifugation. The isolated membranes were enriched 15-fold in brush-border enzyme activities (alkaline phosphatase, γ-glutamyl transpeptidase) compared with whole kidney homogenates and were substantially free of other contaminating membranes. The yield of the BBM preparation was 40{\%}. The BBM vesicular transport of several organic solutes was characterized by a rapid filtration technique at 25°C. D-glucose, p-aminohippurate (PAH), and urate entered the same osmotically active space (2-3 μl/mg protein) and binding was minimal (<20{\%} for PAH). An uptake overshoot for 3-O-methyl-D-glucose (20 μM) by reptilian BBM was observed only in the presence of an inwardly directed NaCl gradient and was abolished by 0.1 mM phlorizin. Reptilian BBM exhibited Na-gradient-stimulated uptake of PAH (90 μM) with an overshoot that was inhibited by other organic acids and by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). In contrast, urate uptake (30 μM) appeared to be Na independent and not appreciably affected by other organic anions or SITS. The presence of specific transport systems for organic solutes in the isolated membrane preparation distinctly characterizes the BBM of reptilian kidney.",
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