Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.     

SPE-HPLC法快速检测发酵液中紫杉醇的含量

针对红豆杉内生真菌发酵液中紫杉醇的含量测定进行探讨,以建立快速高效低耗的检测方法.采用C_(18)固相萃取柱对紫杉醇进行吸附,用不同浓度的甲醇-乙酸铵和甲醇分别作为洗脱剂对其进行洗脱,比较两者的洗脱效果,洗脱液用HPLC进行检测;色谱条件为:流动相甲醇(v):水(v):乙腈(v)=20: 45: 35,流速:0.70 ml/min,检测波长:227 nm.结果表明,浓度为80%的甲醇溶液洗脱效果较好,紫杉醇的回收率为87.6%.     

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. This resistance (> 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture or in suspension or whether choleragen was bound at 0 or 37°C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside G_M1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [³H]G_M1, up to 70% of the cell-associated G_M1 was extracted under the mild conditions. When the G_M1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of G_M1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [³H]galactose by immunoadsorption, only labeled G_M1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.     

para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine

In the oximolysis reaction para-aldoximes K027 and TMB-4 react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4 and 306 M(-1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG#, DeltaH# and DeltaS#, were also determined for oximolysis reaction.     

Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubule-associated protein 2 (MAP 2) from the rat brain was phosphorylated by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. The apparent Km for MAP 2 of Kinase II was 0.2 μ$\text{M}$. The maximum incorporation of phosphate into MAP 2 by the action of Kinase II was about 5 mol of phosphate per mol of MAP 2, while that by the action of cAMP-dependent protein kinase was about 3 mol of phosphate per mol of MAP 2. When microtubule-associated proteins were incubated with both Kinase II and cAMP-dependent protein kinase together, about 7 mol of phosphate were incorporated into 1 mol of MAP 2.     

Enzyme-bound early product of purified poly(ADP-ribose) polymerase.

Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD⁺, ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme.     

Inactivation and reactivation of mitochondrial respiration by charged detergents

Respiration of submitochondrial preparations can be inhibited by the cationic detergent cetyl trimethyl ammonium bromide and the anionic detergent sodium dodecyl sulfate in the range of 0.3–2 μmol of detergent per mg of mitochondrial membrane protein depending on the substrate and detergent used. This inhibition can be rapidly reversed by neutralizing a given detergent by the detergent of the opposite charge. At higher levels of the inhibiting detergent, no such reactivation was observed. Spin labeling assays of membrane structure were used to correlate structural effects with the loss and recovery of respiratory functions.Because the detergents progressively disrupt membrane structure, mitochondria were cross-linked with bifunctional imidoesters to an extent that osmotic properties and detergent lysis were gone, but respiration remained. Such fixed respiring mitochondria also show inhibition reactivation phenomena.     

Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-μl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C₁₈) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 μM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 μM and 14% (n=6 days) at 0.3 μM NTBC in plasma. The quantitation limit was approximately 0.3 μM using 20 μl of plasma.     

Membrane surface potential and the reactivity of the System II primary electron acceptor to charged electron carriers in the medium

A hypothesis is proposed to explain the change in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase.Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149–166).Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.     

Cross-linking of chromosomal non-histone proteins to DNA by UV radiation and some antitumor drugs

Antibodies reacting specifically with HeLa cell chromatin can be elicited by immunization with dehistonized HeLa chromatin preparations. The nature of these chromatin-associated antigens was investigated by cross-linking with UV irradiation or by in vitro exposure of chromatin to 1-methyl-1-nitrosourea (MNU) or 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). With the exception of 1-methyl-1-nitrosourea the described treatment of chromatin (native or dehistonized) significantly increased its immunological reactivity. Dissociation of the chromosomal proteins from DNA by concentrated salt-urea solutions essentially abolished the immunological reactivity of the residual chromatin pellets. The immunological activity was found in the supernatant protein fraction after its reconstitution with purified human placenta DNA. UV irradiation or alkylation of chromatin cross-linked the active proteins to DNA and prevented their dissociation. It is concluded that the immunologically cell-specific antigens in HeLa chromatin exist as closely associated complexes of chromosomal protein(s) with DNA.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

Photodesmosine,an isomer of desmosine obtained by photolysis of this amino acid in ultraviolet light

Desmosine and isodesmosine are two isomers representing the main cross-links of elastin. We describe a new isomer, photodesmosine, which is produced by the photolysis of desmosine at 254 nm. The mechanism of this photolysis is described and is shown to consist of two competing paths. After opening of the pyridium ring to give a tetrasubstituted aminoketone, this compound can either be hydrolysed to give lysine and a trisubstituted analogue of glutaconic aldehyde or undergo a recyclisation and rearomatisation to give a pyridium compound substituted in positions 1, 2, 3 and 4. An understanding of this mechanism is important in order to use photolysis as a specific method to break elastin cross-links. Although only desmosine and isodesmosine have been reported in purified elastin, the chromatographic properties of photodesmosine suggests that if other natural isomers exist in this protein they could be eluted from an ion-exchange resin at much earlier times than those observed in the case of the two already described cross-links.