Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

Summary An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H⁺-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na⁺-K⁺-ATPase (Mayahara et al. 1980) and gastric H⁺-K⁺-ATPase (Fujimoto et al. 1986). K⁺-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The K⁺-independent NPPase activity was diminished by the inhibitors of H⁺-ATPase such as N-ethylmaleimide (NEM) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H⁺-ATPase which plays a role in acidification.     

The effect of sodium chloride on the Ca2+, K+ and Na+ concentrations of the seed coat and embryo of Acacia tortilis and A. coriacea

The uptake of Na⁺ and the loss of Ca²⁺ and K⁺ by seeds of Acacia tortilis (Forsk.) Hayne (salt tolerant) and A. coriacea DC. (salt sensitive) were determined after 24 h soaking in 250 mol m^-1,3 NaCl or in distilled water. Na⁺ uptake was higher by the seed coat than by the embryo of both species and higher by A. coriacea than by A. tortilis. The greater Na⁺ uptake by A. coriacea was associated with greater Ca and K⁺ leakage. The Na⁺ concentration of solution imbibed by the embryo of both species was lower than the Na⁺ concentration in the external solution, indicating an exclusion of Na⁺. When A. tortilis and A. coriacea seeds were treated with a series of NaCl concentration (0–400 mol m^-1,3), the exclusion mechanism was particularly clear with A. tortilis at lower concentrations (50 and 150 mol m^-1,3) of NaCl. In contrast, the seed coat of both species accumulated Na⁺. Thus the seed coat may play an important role in ion exchange. These results show that it is important to consider the seed coat and embryo separately rather than the whole seed when considering ion exchange in relation to salinity tolerance.     

渗透剂对白刺花体细胞胚成熟及萌发的影响

该研究以蔗糖、麦芽糖、山梨醇及PEG(6000)为渗透剂,探讨了不同渗透剂对白刺花体细胞胚发育、胚成熟及萌发的影响。结果表明:白刺花下胚轴形成的胚性愈伤组织接种至MS+2,4-D 0.2 mg·L~(-1)+NAA 1.0 mg·L~(-1)+6-BA 2.0 mg·L~(-1)+TDZ 1.0 mg·L~(-1)+蔗糖40 g·L~(-1)+谷氨酰胺100 mg·L~(-1)+植物凝胶3g·L~(-1)的培养基上,体细胞胚发生率高达66. 21%,总胚数为79个; 7%蔗糖可使体细胞胚成熟率高达64.36%,同时也可提高多子叶畸形胚形成; 2%麦芽糖+2%山梨醇+4%蔗糖组合使体细胞胚成熟率最高达88.89%,畸形胚比例最低; 30 g·L~(-1)PEG培养时,体细胞成熟率最高,为82.35%;鱼雷期的体细胞胚最合适转接,可使体胚萌发率达90.58%,复合糖上培养得到的成熟体细胞胚生根率最高,为87.47%。这为实现白刺花体细胞胚育苗奠定了理论基础,并提供了可行的方案。     

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na⁺ fluxes and spatio-temporal fluctuations of Na⁺ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na⁺-K⁺ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na⁺-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na⁺ -dependent transporters and Na⁺ signalling.     

Prodigiosin 25-C Suppression of Cytotoxic T Cells in Vitro and in Vivo Similar to That of Concanamycin B,a Specific Inhibitor of Vacuolar Type H+-ATPase

The effects of prodigiosin 25-C (PrG) which preferentially suppresses cytotoxic T cells (CTL), was examined in comparison with concanamycin B (CMB), a specific inhibitor of vacuolar type H⁺ -ATPase (V-ATPase). PrG and CMB directly inhibited the cytotoxic function of CTL and neutralized acidic organelles of CTL in vitro. In addition, PrG or CMB was injected in C57BL/6 mice after immunization with an allogeneic mastocytoma, P815. PrG and CMB inhibited the killing activity of CTL against the tumor and reduced the population of CD8⁺ cells without affecting CD4⁺ and B220⁺ populations in the spleen. PrG and CMB had only a negligible effect on antibody production induced by sheep red blood cells (SRBC) and mitogenic responses of lymphocytes. These results suggest that PrG and CMB have similar immunosuppressive properties at least through their inhibitory effects on acidification of intracellular organelles required for the effective function of CTL.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

He-Ne 激光辐照与UV-B 辐射对小麦幼苗细胞器中Na+/K+-ATP 酶活性的影响

分别采用5 mJ.s^-1.mm^-2 He-Ne激光辐照、10.08 kJ.m^-2.d^-1增强UV-B辐射及二者组合对小麦(Triticum aestivum)晋麦8号(Triticum aestivum ‘Jinmai8’)幼苗进行处理。第5 天开始测定各处理小麦幼苗叶片中线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性的变化。结果表明, 随着处理天数的增加, 小麦幼苗叶片线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性均在第6天下降, 第7天升高, 而后又逐渐下降。在处理的第7天, 仅He-Ne激光辐照可使小麦幼苗叶片线粒体、叶绿体及细胞溶质中Na⁺/K⁺-ATP酶活性升高; 增强UV-B辐射使各细胞器中Na⁺/K⁺-ATP酶活性下降; 复合处理后小麦各细胞器中Na⁺/K⁺-ATP酶活性均高于UV-B单独辐射处理。实验结果表明 , 一定剂量的He-Ne激光辐照能够部分修复UV-B辐射对小麦幼苗细胞器中Na^+/K⁺-ATP酶造成的损伤。     

The Potassium Requirement for Growth and Embryogenesis in Wild Carrot Suspension Cultures

A requirement for potassium for growth and forembryogenesis in suspension cultures of wild carrot (Daucus carota L.) was demonstrated. The concentration of K⁺ required for maximal growth (1 mM) was less than that required for maximal embryogenesis (20 mM). Neither Na⁺ nor NH₄⁺ could replace K⁺. Ammonium ion enhanced embryogenesis when K⁺ was present at suboptimal levels greater than 1 mM. Nitrogen sources strongly influenced growth and embryogenesis, but the effects of nitrogen were separable from those of K⁺. Subline differences were noted. Subline CSC-29 produced nearly half the maximum embryo number in 1 mM K⁺ while CSC-31 produced no embryos at that K⁺ concentration. Growth of CSC-29 was slightly repressed by Na⁺, but no more than by similarconcentrations of K⁺. Growth of CSC-31 in 1 mM K⁺ was strongly repressed by Na⁺. Embryogenesis in CSC-29 was unaffected by Na⁺. In CSC-31, Na⁺ repressed embryogenesis at lower concentrations of K⁺.     

Modulation of Serine/Threonine Protein Kinase Activity in Chloramphenicol-Resistant Mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cml^r) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in the early stationary phase. By labeling in vivo, the Cml^r mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cml^r mutant, and a Cml^s revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K proteins as pkn 24, pkn 32, pkn 13, pkn12, and pkn5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca²⁺-dependent PK 85K in the Cml^r mutant. It was assumed that PK 85K is involved in regulating the avermectin production.     

On the role of K+ on succinic dehydrogenase activity

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K⁺ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K⁺ does not changes theK _m for succinate but diminishes theV _max. In addition, the data provide evidence that mitochondria oxidizing glutamatemalate in a sucrose medium show a higher activity of succinate dehydrogenase than mitrochondria incubated in KCl.     

Developmental changes of Islet-1 and its co-localization with pituitary hormones in the pituitary gland of chick embryo by immunohistochemistry

Although Islet-1 expression in the pituitary gland of early mouse embryo has been previously described, there are no reports concerning the correlation of Islet-1 expression with lineage restrictions in cell types at the later stages of pituitary development. The role of Islet-1 in chickens is also unknown. The purpose of this study was to follow, by using immunohistochemistry, the ontogeny of pituitary Islet-1 and the various cell types that contain Islet-1 throughout chick embryo development. A few Islet-1-immunopositive (Islet-1⁺) cells were first detected in the pituitary primordium in two out of six embryos at embryonic day 5.5 (E5.5), most of the Islet-1⁺ cells being ventrally located. As development progressed, many more Islet-1⁺ cells were observed throughout the pars distalis. The relative percentage of Islet-1⁺ cells amongst the total Rathke’s pouch cells was 4.4% at E6.5. This increased significantly, reaching 11.1% by E10.5, followed by no significant change until hatching. Dual immunohistochemistry showed that adrenocorticotrophs, somatotrophs and lactotrophs did not express Islet-1. The cellular types expressing Islet-1 included luteinizing-hormone-positive (LH⁺) gonadotrophs and thyroid-stimulating-hormone-positive (TSH⁺) thyrotrophs. The cells co-expressing LH and Islet-1 were initially detected at E6.5, the proportion of LH⁺ cells possessing Islet-1 being about 4%; this increased to 63% at E14.5, followed by no significant changes until hatching. TSH and Islet-1 co-localized cells were first observed at E10.5, with about 37% TSH⁺ cell expressing Islet-1; this increased to about 50% by E16.5, after which there was no evident change until hatching. These results suggest that Islet-1 is involved in determining the cell lineages, proliferation, differentiation and maintenance of hormone-secreting functions of pituitary gonadotrophs and thyrotrophs of chick embryo. J. Liu and Y. He contributed equally to this article. This work was supported by grants from Beijing Natural Science Foundation (6042013) and the Natural Science Foundation of China (30471264, 30325034).     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

The Role of Acidocalcisomes in Parasitic Protists1

ABSTRACT. Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP_i) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca²⁺‐ATPase, V‐H⁺‐ATPase, H⁺‐PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and channels (aquaporins), while its matrix contains enzymes related to PP_i and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non‐pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP_i and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome‐related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP_i, poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.     

Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis

Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI–EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD⁺/AveD⁻ and AveD⁻/AveD⁺). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23→Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD.     

Fluorescence of proteins in aqueous neutral salt solutions. II. Influence of monovalent cation chlorides, particularly cesium chloride.

The effects of varying concentrations of monovalent cation chlorides on the fluorescence of nine proteins were studied. These are discussed in terms of “direct” or “indirect” interactions with the aromatic amino acid residues. Cs⁺ is the only cation that quenches fluorescence of proteins due to “direct” interaction with aromatic amino acid residues. Quenching is due to collisional processes. An agreement with the Stern-Volmer relationship is shown and the values of [(K_Q)eff] and [(f_a)eff] are calculated. These values confirm that the fraction of fluorescence accessible to Cs⁺ belongs to the “exposed” fluorophors. The mechanism of quenching by Cs⁺ is due to the heavy-atom effect because phosphorescence enhancement is also seen at the same time. The chlorides of Na⁺, K⁺, Rb⁺, NH₄⁺, and Li⁺ do not have a similar effect on the fluorescence of all proteins. For a given protein a gradation of the same effect (i.e., quenching or dequenching) is seen. Interactions with factors that “inderectly” affect fluorescence of any protein are involved and the structural features of the protein are responsible for such “indirect” effects. The results indicate that neutral salts can act in more than one manner. The changes in fluorescence are indicative of electrostatic and lyotropic effects of ions. Only electrostatic interactions which occur in the vicinity of tryptophan in proteins are reflected. Li⁺ shows strong interactions with proteins. In 4 M LiCl, BSA, papain, and trypsin show fluorescence changes that are indicative of changes in protein structure.     

Induction of the Na+/Pi cotransport system in the plasma membrane of Chara corallina requires external Na+ and low levels of Pi

It was shown in previous studies that the giant freshwater alga Chara corallina does not control its Na⁺‐dependent Pi uptake by monitoring the internal Pi concentration and it was hypothesized that Chara may instead detect changes in Pi supply from the environment. The present work investigated the conditions that control the induction and inactivation of high affinity Na⁺/Pi influx in Chara. Withdrawal of Pi from the external medium resulted in a gradual increase in the rate of uptake measured immediately after Pi was resupplied. The increase continued for at least 7 d of starvation. In the initial stages, 0·5 or 1 µm Pi were more effective at inducing transport activity than no Pi, suggesting that low levels of Pi are actually required for induction. The high Na⁺‐dependent Pi uptake observed in Pi‐starved cells was inactivated by treatment with as little as 1 µm Pi over 6 d. External Na⁺ plays a major role in controlling the capacity for Na⁺/Pi cotransport activity, and in the absence of Na⁺, both induction and inactivation were either delayed or abolished. Na⁺ starvation stimulated Na⁺ uptake even though there were no measurable changes in the concentrations of Na⁺, or of K⁺ or Pi in either the vacuole or cytoplasm. It was concluded that both substrate (Pi) and driver ion (Na⁺) are required at adequate concentrations for the induction of the cotransporter. In the case of Pi, it was suggested that passive leakage of Pi from the cell into the apoplast is sufficient for this purpose but that supplementation by up to 1 µm Pi is more effective at the earlier stage. A mechanism for sensing the external supply of Pi is proposed.     

Organelle-localized potassium transport systems in plants

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K⁺ transport systems allowing K⁺ to move across the membrane. K⁺ transport systems in plant organelles act coordinately with the plasma membrane intrinsic K⁺ transport systems to maintain cytosolic K⁺ concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K⁺ channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K⁺ homeostasis of the cytoplasm. The initial electrophysiological measurements of K⁺ transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K⁺ transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K⁺ transport system has been isolated from cyanobacteria, which may add to our understanding of K⁺ flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K⁺ transport proteins.     

Mitochondria from the salt-tolerant yeast <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> (halophilic organelles?)

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na⁺ and 10 mM K⁺; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca²⁺ or Mg²⁺. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K⁺ and Na⁺, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K⁺/Na⁺-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na⁺ or K⁺. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K⁺ and Na⁺ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.