Brush Border Membrane Proteins Research Articles

In vitro phosphorylation of rat kidney proximal tubular brush border membranes.

The phosphorylation of rat renal brush border membrane protein was analyzed after incubation of cortical slices with 32P-orthophosphate and compared with the phosphorylation by gamma-32P-ATP of isolated brush border vesicles. Phosphate incorporation into brush border membranes isolated from slices was linearly related to the incubation time as well as to the specific activity of orthophosphate present during slice incubation. Incorporation of phosphate into proteins reached an equilibrium after about 60 min, whereas incorporation of phosphate into lipids increased continuously. In brush border membranes isolated from slices incubated with orthophosphate (32P), the addition of cAMP or theophylline produced a dephosphorylation of a 47,000-dalton protein; no increased phosphorylation was observed. In brush border membranes, phosphorylated with gamma-32P-ATP, cAMP and dibutyryl cAMP (dB-cAMP) produced an increase in phosphorylation but no dephosphorylation. Sodium-dependent phosphate transport in brush border membranes was not altered by an incubation of slices with cAMP or dB-cAMP. These observations suggest that the phosphorylation machinery of isolated rat renal brush border membranes does not correspond with the mechanisms leading to phosphate incorporation into brush border membrane proteins in the intact cell.

Polyamines stimulate d-glucose transport in isolated renal brush-border membrane vesicles

Polyamines are natural constituents of most living organisms. However, their function in normal or pathological conditions is not fully understood. We have investigated in vitro effects of polyamines on characteristic properties of isolated renal brush-border membrane vesicles in order to determine whether polyamines have a regulatory role in membrane transport processes. The polyamines putrescine, spermidine and spermine were found to stimulate d-glucose uptake. Diffusional l-glucose uptake was not altered, indicating that the polyamines affected the active transport of d-glucose, rather than inducing nonspecific changes in membrane lipid properties. The amiloride-sensitive Na +/H + exchange was slightly inhibited by polyamines while Mg 2+-ATPase activity was stimulated. The polyamine effects could not be explained solely by the polycationic properties of these agents, since polycationic polypeptides had an opposite effect. For example, lysozyme was found to inhibit d-glucose transport. Spermine was incorporated into the trichloroacetic acid-insoluble fraction of brush-border membrane proteins. Results indicated that this incorporation process consisted of at least two components: a Ca 2+-independent component and a Ca 2+-dependent component, possibly as a result of transglutaminase activity which was present in the isolated renal brush-border membranes. By using SDS-polyacrylamide gel electrophoresis in conjunction with fluorography, | 3H|spermine was shown to be incorporated into several brush-border membrane proteins, mainly the 57 kDa, 74 kDa, 100kDa, a heavy molecular weight band (greater than 200 kDa) and a low molecular weight band (less than 10 kDa). Our results suggest that the polyamine effects on membrane function may be due to a covalent modification of membrane proteins, possibly via a transglutaminase-mediated incorporation of polyamines or to the crosslinking of membrane proteins.

Purification and characterization of kidney and intestinal brush-border membrane trehalases from the rabbit

Trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) was solubilized from the brush-border membrane of rabbit intestine and kidney by Emulphogen BC 720. The intestinal and kidney enzymes were purified 10 400-times and 4457-times respectively, in a five-step procedure, including DEAE-Trisacyl, chromatofocusing, AcA 34 gel filtration, HA Ultrogel and preparative polyacrylamide gel electrophoresis. The purified enzymes were homogeneous on polyacrylamide gel electrophoresis. The specific activities of kidney and intestinal pure trehalase were identical. Kidney and intestinal trehalases have the same molecular weight (about 85 000 by Sephadex G-200 gel filtration and 75 000 by SDS-polyacrylamide gel electrophoresis). A Stokes radius of 38 Å was determined. Detergent solubilized trehalase is not retarded phenyl-Sepharose CL-4B chromatography. The isoelectric point, measured by chromatofocusing, is between pH 3.8 and 4.2 for kidney trehalase and between pH 4.6 and 4.8 for intestinal trehalase. The enzymic properties for both kidney and intestinal trehalases are identical. The optimum pH is between 5.5 and 6.0. Trehalase is heat stable up to 50°C. The apparent K m was found to be 3.5 mM in maleate buffer pH 6.0. The activation energy of trehalase is 11.17 kcal/mol. Tris, sucrose and phloridzin are fully competitive inhibitors with K i of 3.7 mM, 3.1 mM, 1.1 mM respectively. Schiff's staining on polyacrylamide gel and interaction with Con A-Sepharose indicate that trehalase is a glycoprotein. Trehalase accounts for 0.1% and 0.3% of total brush-border membrane protein of intestine and kidney, respectively.

Chromatofocussing and centrifugal reconstitution as tools for the separation and characterization of the Na +-cotransport systems of the brush-border membrane

Chromatofocussing was used for the separation of brush-border membrane proteins from calf kidney into 4 or 5 fractions over the pH range 4.0 to 7.4. These fractions were reconstituted into proteoliposomes by gradient centrifugation. Determination of the sodium-dependent solute uptake by proteoliposomes reconstituted from different chromatofocussed fractions showed that the sodium- d-glucose cotransport system was present in the fraction eluted between pH 5.3 and 5.8, and that the sodium-phosphate cotransport was present in fractions eluted between pH 4.6 and 5.3 and between pH 5.8 and 6.6, sodium-alanine cotransport could be detected in almost all fractions. Marker enzymes of the brush-border membrane, such as alkaline phosphatase. γ-glutamyltransferase and aminopeptidase M etc. were also found to be eluted at pH 7.0–7.4, 4.0–4.1 and 5.6–5.8, respectively. These results suggest that chromatofocussing is a promising tool for the separation membrane proteins and for pre-purification of the sodium- d-glucose cotransport system. It can be further concluded that the sodium-dependent phosphate transport across the brush-border membrane is not dependent upon alkaline phosphatase activity.

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles.

The effects of intravesicular NAD on Na+-dependent 32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis. It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited. 32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of 3H-adenine could be detected. The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.

Role of c-AMP and NAD in the regulation of Na+-dependent phosphate transport across rat renal brush border membranes.

The activity of the sodium-dependent Pi-transport system in the brush border membrane changes in concordance with the tubular phosphate reabsorption under a variety of physiological conditions (Dousa and Kempson, 1982). Two mechanisms were postulated to be involved in these adaptations: a cyclic-AMP dependent phosphorylation of brush border membrane protein(s) (Hammerman and Hruska, 1982) and a direct interaction of NAD with the membrane (Kempson et al., 1981), apparently via a ADP-ribosylation of certain membrane proteins (Hammerman et al., 1982).

NAD+-dependent ADP-ribosyltransferase in renal brush-border membranes.

Oxidized nicotinamide adenine dinucleotide (NAD+) in cytosol may interact with renal brush-border membranes (BBM) and inhibit BBM phosphate transport. The possible mechanism of interaction was investigated in the present study. Incubation of BBM with [adenine-3H]NAD+ led to acid-stable binding of 3H to the BBM, in contrast there was no binding of 14C when [carbonyl-14C]NAD+ was used. The data are consistent with an ADP-ribosylation mechanism involving transfer of ADP-ribose from NAD+ to BBM. This was confirmed by using [adenylate-32P]NAD+ and by the release of bound 32P in the form of 5'-[32P]AMP when the BBM were treated with snake venom phosphodiesterase. After gradient centrifugation of BBM the ADP-ribosyltransferase was recovered at the same density as known BBM enzymes, indicating that ADP-ribosyltransferase is an intrinsic BBM component and not a contaminant. These findings indicate that cytosolic NAD+ may be used for ADP-ribosylation of BBM proteins and that this may be a mechanism for regulating the BBM phosphate transport system.

Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase. 5′-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [ adenine-2,8- 3H]NAD +, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20°C, the equilibrium binding was obtained at 15 min, while NAD binding at 0°C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20°C. Brush border membrane inactivated by heating at 95°C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both α-NAD and β-NAD stereoisomers displaced bound [ 3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD +. Adenine, nicotinamide, pyrophosphate, of 5′-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10 −4 M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site ( K d = 1.9·10 −5 M) and a low-affinity binding site ( K d = 2.2·10 −3 M). (4) Unlike concentrative uptake of d-[ 14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na o + > Na i +], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike d-[ 14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.

Characterization of phosphate binding by alkaline phosphatase in rat kidney brush border membrane.

Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by gamma-32P ATP at 0 degree C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1 +/- 5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein. Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparent Km: one at 40 microM for low and one at 390 microM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.

Vitamin D. Its effect on the protein composition and core material structure of the chick intestinal brush-border membrane.

Vitamin D3 is known to stimulate the absorption of calcium across the asymmetric intestinal epithelial cells. Efforts to elucidate the mechanism of stimulation of intestinal calcium transport by vitamin D are now focused on evaluating the protein composition and topology of the brush-border membrane and its associated core material. Intestinal brush-border membranes were isolated from vitamin D-replete and vitamin D-deficient chicks. Core material proteins were isolated, by sedimentation, from brush-border membranes which were solubilized with Triton X-100. As determined by polyacrylamide gel electrophoresis, dietary vitamin D3 treatment caused no change in the relative amounts of five major core material proteins with Mr = 101,000, 94,000, 67,000, 42,000 (actin), and 17,000. In contrast, dietary vitamin D3 treatment caused a significant reduction in the levels of two proteins with Mr = 111,000 (sucrase) and 83,000, and an increase in the levels of a protein with Mr = 78,000 (possibly a subunit of alkaline phosphatase). The Mr = 111,000, 83,000, and 78,000 proteins are readily solubilized by Triton X-100 and are located on the extracellular surface of the brush-border membrane, as judged by [125I]diazoiodosulfanilic acid and lactoperoxidase 125I labeling. A significant vitamin D-dependent difference was found with respect to iodination of isolated core material as evidenced by the 125I labeling patterns of the Mr = 42,000 protein (actin). The Mr = 42,000 protein was labeled two to three times more extensively when associated with core material derived from vitamin D-deficient chicks as compared to vitamin D-replete chicks. Increasing the salt concentration (0-125 mM KCl) present during core material isolation from either vitamin D-replete or vitamin D-deficient chicks yields core material actin which is more susceptible to iodination by both [125I]diazoiodosulfanilic acid and lactoperoxidase. This increase in the extent of actin iodination is coupled to a salt-induced decrease in the stability of the core material which is evidenced by a decrease in the percentage of total brush-border membrane actin which is Triton-insoluble. This strongly suggests that the vitamin D-induced decrease in the accessibility of actin to iodination reagents results from a vitamin D-dependent change in the structure of the core material. Collectively, these results implicate a role for dietary vitamin D3 in maintaining a specified composition and topology of both the brush-border membrane proteins as well as its associated cytoskeletal core proteins, which is possibly important for intestinal calcium transport.

NAD +-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD + hydrolysis

We previously demonstrated inhibition of Na +-dependent 32P i transport in canine renal brush-border membranes in association with NAD +-induced ADP ribosylation of membrane protein(s) and postulated that NAD + inhibits P i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD + resulted in release of P i which was prevented by EDTA. It was proposed that NAD +-mediated inhibition of 32P i transport might occur through this mechanism. To determine whether NAD + inhibited 32P i transport by a mechanism other than or in addition to release of P i, we compared Na +-dependent 32P i counterflow in brush-border membrane equilibrated with P i or with P i generated from NAD +. Release of P i from NAD + incubated with brush-border membrane was confirmed. The increased uptake of 32P i which was demonstrated in brush-border membrane equilibrated with P i was not measured when intravesicular P i was generated from a concentration of NAD + which effected ADP-ribosylation of brush border membranes (100 μM NAD +). In contrast, increased uptake of 32P i was demonstrated when intravesicular P i was generated from 1 μM NAD + which did not effect ADP ribosylation. Mg 2+-dependent ADP ribosylation of brush-border membrane incubated with NAD + was demonstrated which persisted during the time interval of 32P i uptake measurements. Our findings are compatible with the hypothesis that NAD +-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg 2+ and decreased ADP ribosylation.

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [ 32P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ- 32P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ- 32P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.

Cyclic AMP-dependent protein phosphorylation and dephosphorylation alter phosphate transport in canine renal brush border vesicles

We have previously demonstrated that the cAMP-dependent phosphorylation of two or more proteins (bands IX and V) in membrane vesicles isolated from the renal brush border from kidneys of dogs, was associated with decreased Na + -dependent P i transport. In the present studies a specific dephosphorylation of band IX was demonstrated in bursh border vesicles incubated in the absence of F − which had been used in previous studies to inhibit phosphoprotein activity. Dephosphorylation of band IX was 80% complete after 5 min of incubation at which time inhibition of P i transport in membrane vesicles which had been phosphorylated in the presence of cAMP could no longer be demonstrated. Dephosphorylation of band IX was no different in vesicles from kidneys originating from parathyroidectomized dogs prior to or following the administration of parathyroid hormone in vivo and normal dogs. We conclude that the cAMP -dependent phosphorylation of brush border membrane proteins may mediate a phosphaturic effect of the hormone. Parathyroid hormone-induced phosphaturia may be terminated through the actioin of a specific membrane phosphoprotein-phosphatase.

Use of monoclonal antibodies in the study of intestinal structure and function.

The hybridoma technique, originally developed by G. Kohler & C. Milstein, is a powerful new experimental approach for analysis of complex biological systems, and is particularly suited for identification and study of surface-membrane antigens. This technique has been used for the production of monoclonal antibodies to intestinal brush border membrane proteins. Spleen cells, obtained from BALB/c mice immunized with purified brush border membranes, were fused with NSI mouse myeloma cells, and hybrids were selected with a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Hybridoma cultures were screened for production of specific antibodies by radio-immunobinding assays and by immunofluorescent staining of intestinal frozen sections. Selected hybridoma cultures were cloned twice and used for the production of large amounts of antibodies, which were characterized. Nineteen monoclonal antibodies have been prepared to date, about half of them specifically staining the brush border membrane of mature enterocytes. Ten of the antibodies specifically immunoprecipitate surface-membrane proteins, which were analysed by sodium dodecyl sulphate slab-gel electrophoresis, by two-dimensional slab-gel electrophoresis, and by specific enzyme assays. Two antibodies were found to be specific for sucrase-isomaltase, one for an aminopeptidase, two for an isoenzyme of alkaline phosphatase that is present exclusively in the proximal small intestine, and one for maltase-glucoamylase. These monoclonal antibodies, and others prepared by similar techniques from mice immunized with a wide variety of intestinal subcellular fractions, should prove invaluable tools for the study of the biosynthesis of cell-surface proteins, the fetal and postnatal development of specific intestinal functions, and the process of cell differentiation in the intestinal epithelium.

The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border.

Purified brush border fractions prepared from rat kidneys were solubilized in detergent, iodinated, and subjected to immunoprecipitation to identify the pathogenic antigen present in brush border membranes that is responsible for the production of Heymann nephritis (HN). Purified IgG prepared from the sera of rabbits or rats immunized with a crude cortical preparation, known as Fx1A, precipitated multiple peptides, whereas IgG eluted from glomeruli of rats with active or passive HN specifically immunoprecipitated a single large glycoprotein (Mr = 330,000). This protein (gp330) was subsequently purified by gel filtration and lentil lectin affinity chromatography from detergent-solubilized brush border membranes. When rats were immunized with purified gp330, they developed anti-brush border antibodies and active HN. IgG prepared from the serum of rats with active HN caused passive HN when injected into normal recipients. Rats immunized against brush border membrane proteins depleted of gp330 developed anti-brush border antibodies but did not develop HN. Further analysis of gp330 indicated that it is solubilized by detergent treatment of isolated brush border microvilli, and its antigenic component is released from intact microvilli by trypsin. By immunoperoxidase staining it was localized to the luminal side of the brush border membranes. These results indicate that (i) gp330 is the pathogenic antigen of HN; (ii) the antigen is a glycoprotein of the brush border membrane; and (iii) it is disposed with its pathogenic domain(s) facing the tubule lumen.

Immunochemical analysis of high molecular mass urinary proteins

Urinary high molecular mass proteins (fraction P) solubilized in Triton X-100 and by papain have been compared with the solubilized human renal brush border membrane proteins. Crossed immunoelectrophoresis of Triton X-100 fraction P extract, by means of two polyspecific antisera directed against either renal membrane or fraction P, revealed eleven immunoprecipitates antigenically identical with detergent renal membrane antigens. Among them, five hydrolases were identified by zymogram staining: microvillus aminopeptidase, maltase, trehalase, γ-glutamyltransferase and alkaline phosphatase. Eight papain-solublized fraction P proteins and Triton X-100-solubilized membrane extract presented ‘identity’ patterns in tandem crossed immunoelectrophoresis, but differed in their amphiphilicity, as demonstrated by the change of precipitation pattern on charge-shift crossed immunoelectrophoresis. Among the eleven detergent-solubilized fraction P antigens, nine were proved to be amphiphilic proteins and six presented bidirectional charge shifting properties similar to those of renal membrane antigens. Quantitatively, five detergent fraction P proteins were found in the same amounts as in renal membrane extract, two in lesser amounts and four in greater. Moreover, the same two plasma proteins were identified in fraction P as in the renal membrane. Thus important similarities exist between the urinary fraction P and the native renal membrane.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns.

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 +/- 0.9) as compared with scrapings of intestinal mucosa (14.8 +/- 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase-glucoamylase-isomaltase (band 4), alkaline phosphatase (bands 9-10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.

Studies on the incorporation of radioactive amino acids into tissue and brush border membrane proteins of mouse jejunum in organ culture

1. 1. The incorporation of radioactive amino acids into brush border membrane proteins of mouse jejunal mucosa has been investigated in organ culture and compared to similar data obtained in intestinal culture of other species. 2. 2. Amino acid precurosor pools for protein synthesis remained almost unchanged during 24 hr culture. 3. 3. By pulse experiments, it has been possible to show a constant rate of protein synthesis up to 24 hr culture. 4. 4. Continuous labelling studies have shown a non-linear incorporation of amino acids into tissue proteins, brush border membrane proteins and total proteins (explants + media) synthesized during the culture. A turnover of tissue proteins has been assumed from the release of labelled proteins into the culture media. The analysis of radioelectrophoregrams of brush border membrane proteins and proteins from the particulate fraction of media showed that the same brush border membrane proteins were synthesized in the tissue and released in the media all along the culture. 5. 6. However, some quantitative differences have been found in the relative labelling between protein bands, suggesting that some proteins would be synthesized or released at different rates depending upon the duration of the culture.

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport.

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.