Transforming activity and the level of Tax protein: effect of one point mutation in HTLV-I tax gene.

The transcriptional trans-activator molecule of HTLV-I, Tax, is known to transform rodent fibroblasts. A revertant clone expressing Tax was obtained by treating transformed Rat-I cells harboring a single copy of the tax gene with a mutagen. Sequence analysis of the tax gene of the revertant clone revealed that it had one point mutation at codon 12(CTT----TTT), resulting in a change from Leu to Phe. The colony-forming efficiencies of the cells transfected with the mutant tax gene (mu71 tax) were significantly lower than those transfected with the wild-type by the soft-agar method. This difference was shown to be due to the instability of mu71 Tax.

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.

Expression, localization, and function of an N-terminal half fragment of the rat Na,K-ATPase beta-subunit in HeLa cells.

The N-terminal half of the beta-subunit of rat brain Na,K-ATPase was expressed in HeLa cells transfected with the plasmid pSV2TKneo beta N containing the truncated beta-subunit cDNA to study the assembly and transport of alpha-beta complex. Immunoprecipitation from extracts of metabolically labeled transformed cells demonstrated that the truncated beta-subunit polypeptide (beta N) was neither transported to the plasma membrane nor assembled into an alpha-beta complex with the endogenous alpha-subunit. Cell fractionation experiments showed that the beta N truncated subunit remained unassembled within rough microsomes, suggesting that it never exited from the endoplasmic reticulum (ER). The assembly of the endogenous alpha-and beta-subunits in the beta N-expressing cells was significantly inhibited compared with control cells or with the transformants that did not express the beta N. These results suggest that the N-terminal portion of the beta-subunit interferes with the normal assembly of the endogenous complex which normally takes place in the ER.

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland.

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells.

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.

The (Na+, K+)ATPase of rat kidney: purification, biosynthesis, and processing.

(Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A- and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests. The endoplasmic reticulum (ER)-rich, and Golgi-rich subfractions were prepared from the rat kidney microsomal fraction by sucrose density gradient centrifugation. On the immunoblot, the molecular weight of the alpha subunit in both fractions was 95 kilodalton (Kd); whereas, that of the beta subunit was 50 Kd in the ER-rich fraction and 54 Kd in the Golgi-rich fraction. When treated with endoglucosidase H, the 50 Kd component was converted to 38 Kd, but the 54 Kd component was endoglucosidase H resistant. These results suggest that the beta subunit (38 Kd) is glycosylated cotranslationally in the ER (50 Kd) then is converted to the mature type subunit (54 Kd) in the Golgi apparatus.