Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter.

Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K+-Cl- cotransporter1 (KCC1). We have referred to the predicted protein as K+-Cl- cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potential N-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor alpha, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of 86Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl- but not Na+ and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.

Purification of a novel endothelin-converting enzyme specific for big endothelin-3.

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.

Purification of insoluble nitric oxide synthase from rat cerebellum.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.

Purification of nitric oxide synthase from rat macrophages.

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).

Cytosolic and membrane-bound nitric oxide synthase.

Cytotoxic activated macrophages were sonicated and centrifuged. The activity of nitric oxide (NO) synthase was present in the supernatant and independent of Ca2+. The pellets were washed three times and treated with buffer containing 0.1% Triton X-100 or buffer alone, followed by centrifugation. The supernatant containing Triton X-100 showed NO synthase activity that was dependent on Ca2+, whereas the supernatant without the detergent had little activity. These data suggest that there are two forms of NO synthase: cytosolic and membrane-bound enzymes.

Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils.

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.

Stabilizing factor(s) of nitric oxide (NO) synthetase.

We found that the cytosol of rat peritoneal polymorphonuclear neutrophils contains factor(s) that can stabilize an unstable enzyme, nitric oxide synthetase, in the cytosol. This enzyme has been purified to a single protein from the cytosol. Its half-life was 3 hours at 4 degrees C and was prolonged to greater than 24 hours by the stabilizing factor in the cytosol. The molecular weight of the stabilizing factor was greater than 100,000. Its activity was lost by the treatment with heating or alkali for 1 min or with acid for 5 min. It did not adhere to the carboxymethyl or diethylaminoethyl column at neutral pH. This stabilizing factor(s) may play a role in the regulation of the nitric oxide synthetase.