Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.

Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes

Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na(+)-K(+)-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was approximately 20 pmol.min-1.oocyte-1 in the presence of blockers of Na(+)-K(+)-ATPase and conductive K+ transport. Bumetanide (10 microM) inhibited > 90% of this uptake, suggesting involvement of Na(+)-K(+)-2Cl- cotransport. This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide [50% inhibitory concentration (IC50), 2 x 10(-11) M] > bumetanide (IC50, 7 x 10(-8) M) > furosemide (IC50, 2.5 x 10(-6) M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by approximately 75%, whereas a doubling of medium osmolarity increased it by approximately 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an approximately 140-kDa oocyte membrane protein. These results demonstrated that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine cDNAs coding for the centrosomal antigen centrosomin A

Screening of an induced Ehrlich ascites cell-derived lambda gt11 cDNA library with an antibody (GP1), immunoreacting specifically with centrosomal antigen(s) of interphase and mitotic cells of different species, released a partial cDNA clone (lambda P10A) encoding the carboxy-terminal section of a centrosome-specific antigen. This specificity of the clone lambda P10A could be verified by lacZ-directed antigen expression from Escherichia coli Y1089 lysogenized with the recombinant phage lambda P10A and subsequent production of centrosome-specific antibodies by means of the recombinant antigen. Using the lambda P10A insert as a probe, two types of cDNA clones were identified in a lambda gt10 cDNA library by plaque-hybridization. The inserts of PN1 type clones were 1.2 kb (kilobases) and those of PN5 type clones were 2.2 kb in length. The DNA sequence of a PN1 type clone revealed its full-length cDNA nature. The open reading frame of PN1 encodes a rather hydrophilic and highly charged 34.5 × 10(3) Mr polypeptide comprising short but apparently significant strings of 100% sequence identity with the major nuclear lamina polypeptides lamins A/C and lamin B. Restriction enzyme mapping of PN1 and PN5 inserts, cross-hybridization experiments and comparison of overlapping DNA sequences indicate that the 1.2 kb and 2.2 kb cDNAs code for the same 34.5 × 10(3) Mr polypeptide, termed centrosomin A. Western blots of Ehrlich ascites cell proteins show a second, larger GP1 antigen (centrosomin B) whose cDNA has not been cloned. It remains to be investigated whether centrosomin B is encoded by a second mRNA or whether it reflects an oligomeric or a postranslationally modified form of centrosomin A.