The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[³H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [³H]Adenosine derived from 5′-[³H]AMP hydrolysis was further transformed into [³H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [³H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

丁酸钠和pH值对离体蚕豆花药中小孢子有丝分裂的影响

含单核中后期至后期花粉的蚕豆离体花药,经过2mM丁酸钠24小时预处理后,再漂浮培养于pH5.8或7.0的液体培养基。以不经预处理的作为对照。结果如下: 1.培养后9天内,丁酸钠预处理和培养基pH值并不明显影响花粉退化百分率。2.培养初期,pH7.0显著促进小孢子不等分裂。3.丁酸钠预处理抑制培养初期的小孢子有丝分裂,而后又显著增加小孢子均等分裂百分率。4.丁酸钠预处理导致小孢子有丝分裂类型的趋向改变。本文还对丁酸钠导致有丝分裂类型趋向改变的可能原因,进行了讨论。     

The preservation of Mycoplasma capricolum cell intactness after phospholipase A2 treatment

(1) By treating Mycoplasma capricolum cells with phospholipase A₂ about 80% of membrane phospholipids were rapidly hydrolyzed. The rate and extent of hydrolysis (at 37°C) were the same in intact cells and in isolated unsealed membranes. (2) Due to the low endogenous lysophospholipase activity detected in M. capricolum, phospholipase A₂ treatment resulted in the accumulation of lysophospholipids and free fatty acids. The free fatty acids were efficiently extracted from the cells by 1% bovine serum albumin whereas the lysophospholipids were almost fully retained within the cell membrane. (3) Following phospholipase A₂ treatment in the presence of 1% bovine serum albumin, cell intactness was preserved as indicated by the constant absorbance of the cell suspension and the retention of nucleic acids and NADH dehydrogenase activity within the cells. The treated cells showed, however, a slight decrease in K⁺ content and a decrease in cell viability. Viability was fully preserved after phospholipase A₂ treatment of cells grown with exogenous sphingomyelin. (4) Adapting M. capricolum to a cholesterol-poor medium resulted in a marked decrease in the cholesterol to phospholipid molar ratio (from about 1.1 to 0.3). Phospholipase A₂ treatment of the cholesterol-poor cells resuted in cell lysis. Cell lysis was induced in the cholesterol-rich cells by hydrolysing the lysophospholipids accumulated following phospholipase A₂ treatment. (5) It is suggested that after phospholipase A₂ treatment of M. capricolum cells, a relatively stable cell membrane is maintained and cell intactness is preseved due to the interaction of cholesterol, present in high amount in this membrane, with the lysophospholipids formed.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Energized transport of potassium ions in the absence of valinomycin by cytochrome c oxidase-reconstituted vesicles

Valinomycin-independent energized uptake of K⁺ was observed in cytochrome c oxidase reconstituted proteoliposome. The rate of K⁺ influx was proportoinal to the magnitude of electron flux. The energized uptake of K⁺ was abolished by p-trifluoromethoxycarbonylcyanide phenylhydrazone or by nigericin. Using the safranine fluorescence technique, it was demonstrated that even in the absence of valinomycin, liposomes and proteoliposomes reconstituted with cytochrome c oxidase are able to discriminate between Na⁺ and K⁺ and show a preference for K⁺ in the presence of excess Na⁺.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.