Colonic H-K-ATPase beta -subunit: identification in apical membranes and regulation by dietary K depletion

P-type ATPasesrequire both - and -subunits for functionalactivity. Although an -subunit for colonic apical membraneH-K-ATPase (HKc) has been identified and studied, its -subunithas not been identified. We cloned putative -subunit rat colonicH-K-ATPase (HKc) cDNA that encodes a 279-amino-acid protein with asingle transmembrane domain and sequence homology to other rat-subunits. Northern blot analysis demonstrates that this HKc isexpressed in several rat tissues, including distal and proximal colon,and is highly expressed in testis and lung. HKc mRNA abundance is upregulated threefold compared with normal in distal colon but notproximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase ₁ mRNA abundance isunaltered in distal colon of K-depleted rats. Na depletion, which alsostimulates active K absorption in distal colon, does not increaseHKc mRNA abundance. Western blot analyses using a polyclonalantibody raised to a glutathioneS-transferase-HKc fusion proteinestablished expression of a 45-kDa HKc protein in both apical andbasolateral membranes of rat distal colon, but K depletion increasedHKc protein expression only in apical membranes. Physicalassociation between HKc and HKc proteins was demonstrated byWestern blot analysis performed with HKc antibody onimmunoprecipitate of apical membranes of rat distal colon and HKcantibody. Tissue-specific upregulation of this -subunit mRNA inresponse to K depletion, localization of its protein, its upregulationby K depletion in apical membranes of distal colon, and its physicalassociation with HKc protein provide compelling evidence that HKcis the putative -subunit of colonic H-K-ATPase.     

Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.     

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.     

Hepatic progenitor cells

Liver diseases are associated with a marked reduction in the viable mass of hepatocytes. The most severe cases of liver disease (liver failure) are treated by orthotopic liver transplantation. One alternative to whole organ transplantation for patients with hepatic failure (and hereditary liver disease) is hepatocyte transplantation. However, there is a serious limitation to the treatment of liver diseases either by whole organ or hepatocyte transplantation, and that is the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of cells for transplantation have been proposed including embryonic stem cells, immortalised liver cells and differentiated cells. One other source of cells for transplantation found in the adult liver is the progeny of stem cells. These cells are termed hepatic progenitor cells (HPCs). The therapeutic potential of HPCs lies in their ability to proliferate and differentiate into hepatocytes and cholangiocytes. However, using HPCs as a cell therapy cannot be exploited fully until the mechanisms governing hepatocyte differentiation are elucidated. Here, we discuss the fundamental cellular and molecular elements required for HPC differentiation to hepatocytes.     

Basolateral K-Cl cotransporter regulates colonic potassium absorption in potassium depletion

Active potassium absorption in the rat distal colon is electroneutral, Na(+)-independent, partially chloride-dependent, and energized by an apical membrane H,K-ATPase. Both dietary sodium and dietary potassium depletion substantially increase active potassium absorption. We have recently reported that sodium depletion up-regulates H,K-ATPase alpha-subunit mRNA and protein expression, whereas potassium depletion up-regulates H,K-ATPase beta-subunit mRNA and protein expression. Because overall potassium absorption is non-conductive, K-Cl cotransport (KCC) at the basolateral membrane may also be involved in potassium absorption. Although KCC1 has not been cloned from the colon, we established, in Northern blot analysis with mRNA from the rat distal colon using rabbit kidney KCC1 cDNA as a probe, the presence of an expected size mRNA in the rat colon. This KCC1 mRNA is substantially increased by potassium depletion but only minimally by sodium depletion. KCC1-specific antibody identified a 155-kDa protein in rat colonic basolateral membrane. Potassium depletion but not sodium depletion resulted in an increase in KCC1 protein expression in basolateral membrane. The increase of colonic KCC1 mRNA abundance and KCC1 protein expression in potassium depletion of the rat colonic basolateral membrane suggests that K-Cl cotransporter: 1) is involved in transepithelial potassium absorption and 2) regulates the increase in potassium absorption induced by dietary potassium depletion. We conclude that active potassium absorption in the rat distal colon involves the coordinated regulation of both apical membrane H,K-ATPase and basolateral membrane KCC1 protein.     

Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.

Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

Regulation of DRA and AE1 in rat colon by dietary Na depletion

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.     

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase

Active K absorption in the rat distal colon is energizedby an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase -subunit (HKc) has been cloned and characterized (together with the -subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive⁸⁶Rb uptake. In contrast, HKc, when expressed in Sf9cells without a -subunit, yielded evidence of ouabain-insensitiveH-K-ATPase. Because a -subunit (HKc) has recently been clonedfrom rat colon, this present study was initiated to determine whetherH-K-ATPase and its sensitivity to ouabain are expressed when these twosubunits (HKc and HKc) are transfected into a mammalian cellexpression system. Transfection of HEK-293 cells with HKc and HKccDNAs resulted in the expression of HKc and HKc proteins andtheir delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was1) saturable as a function of increasing K concentration with aK_m for K of 0.63 mM; 2) inhibited byorthovanadate; and 3) insensitive to both ouabain andSch-28080. In parallel transfection studies with HKc and Na-K-ATPase1 cDNAs and with HKc cDNA alone, there was expression ofouabain-insensitive H-K-ATPase activity that was 60% and 21% of thatin HKc/HKc cDNA transfected cells, respectively. Ouabain-insensitive ⁸⁶Rb uptake was also identified incells transfected with HKc and HKc cDNAs. These studies establishthat HKc cDNA with HKc cDNA express ouabain-insensitiveH-K-ATPase similar to that identified in rat distal colon.