Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro.

Although calnexin is thought to function as a molecular chaperone for glycoproteins, a prevalent view is that it cannot distinguish between protein conformational states, binding solely through its lectin site to monoglucosylated oligosaccharides. Using purified components in vitro, calnexin effectively prevented the aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also proteins lacking N-glycans, an effect enhanced by ATP. It also suppressed the thermal denaturation of nonglycosylated proteins and enhanced their refolding in conjunction with other cellular components. Calnexin formed stable complexes with unfolded conformers of these proteins but not with the native molecules. Therefore, in addition to being a lectin, calnexin functions as a bona fide molecular chaperone capable of interacting with polypeptide segments of folding glycoproteins.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

Marked expression of glutathione S-transferase A4-4 detoxifying 4-hydroxy-2(E)-nonenal in the skin of rats irradiated by ultraviolet B-band light (UVB).

Enzyme, Western blot, and immunohistochemical analyses indicated that rat skin cytosol contained no detectable level of the homodimeric, alpha-class glutathione S-transferase (rGST) A4-4 which catalyzes the GSH conjugation of the toxic product, 4-hydroxy-2(E)-nonenal (HNE), nonenzymatically formed from n-6 polyunsaturated fatty acid residues of lipids by lipid peroxidation. Rats irradiated by single doses (4000-24,000 mJ/cm(2)) of ultraviolet B-band light (UVB, 200 mJ/cm(2)/min) markedly expressed rGSTA4-4 in the skin at a level one-fifth that of the liver in apparent specific activity toward HNE at a single dose of 24,000 mJ/cm(2). Skin rGSTA4-4 was isolated, purified to homogeneity, and identified with hepatic rGSTA4-4 by reverse-phase partition HPLC and by amino acid sequence analysis of its CNBr fission peptides. Immunohistochemistry with polyclonal antibody raised against rGSTA4-4 demonstrated the selective expression of rGSTA4-4 in epidermis and sebaceous glands localized in dermis after UVB irradiation.     

Polyphosphates in Intraradical and Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus, Gigaspora margarita

The amount of polyphosphate in the intraradical and extraradical hyphae of Gigaspora margarita was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.     

Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.     

Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism.

Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R = 19.2% (R(free) = 23.5%) for the diffraction data at 1.6-A resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys(50) and Cys(124) joins the beta1 strand and the alpha7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.