Cytoskeletal proteins associated with cell surface envelopes from Sarcoma 180 ascites tumor cells

Membrane envelopes prepared from Zn⁺⁺-treated Sarcoma 180 cells contain polypeptides which appear to be related to the putative cellular cytoskeletal elements responsible for control of cell shape and motility. These include actin, myosin, α-actinin and a large polypeptide (mol wt 250,000) with some similarities to spectrin of the erythrocyte membrane. If the envelopes are vesiculated by extraction with alkaline EDTA solutions at low ionic strength, four major polypeptides are released, including the actin and spectrin-like materials; myosin is not extracted. The stabilized envelopes offer a useful source of material for the characterization of cytoskeletal elements and for the investigation of their associations with the membrane.     

Interactions of cytoskeletal elements with the plasma membrane of Sarcoma 180 ascites tumor cells

The association of cytoskeletal proteins with cell surface envelopes from Sarcoma 180 ascites cells has been studied by several techniques previously used successfully in studying the interaction of spectrin with erythrocyte membranes. By electron microscopy the envelopes exhibit irregular exterior surfaces and the presence of substantial amounts of “fuzz” at the interior surface. Extraction of the envelopes at low ionic strength and alkaline pH fragments the membranes and depletes them of the “fuzz” with concomitant elution of four major polypeptides of mol. wt >300 000 (Band E), 250 000, 100 000 and 43 000 D. The last three of these have been tentatively identified as actin-binding protein (ABP), α-actinin and actin. Membrane-associated myosin is not eluted under these conditions. Neither actin nor myosin is eluted under conditions commonly used to depolymerize them. However, myosin can be eluted at high salt concentrations if the envelopes have been previously extracted and fragmented with alkaline buffer as above. Extraction of the envelopes with Triton X-100 removes 60% of the membrane lipid and 70–80% of lactoperoxidase-iodinated cell surface proteins without removal of significant amounts of the cytoskeletal proteins. The Triton residues maintain the shape of the original envelopes but have lost the trilaminar membrane structure. Proteolysis of intact envelopes with trypsin or papain cleaves the high molecular weight polypeptides in the order E > ABP > myosin. Fragmentation occurs with cleavage of E or ABP, but does not appear to require cleavage of myosin. Actin and α-actinin are not appreciably cleaved when associated with the membrane. The results, combined with previous observations, suggest an extensive complex of cytoskeletal proteins attached to the membrane interior surface.     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. II. Kinetics and inhibition studies

Kinetic and inhibition studies of partially purified deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria were performed, including reaction properties, specificity of phosphate donors, phosphate acceptors, influence of nucleotides, nucleosides and their derivatives. The results were compared with those obtained from partially purified AdR kinase from rat liver cytosol.     

小鼠S180腹水瘤细胞在细胞生物学实验教学中的应用及注意问题

摘要细胞生物学实验教学中,经常需要使用大量相同的细胞样品。通过教学实践,作者发现小鼠S180腹水瘤细胞可作为多个实验的细胞材料。该文阐述了小鼠S180腹水瘤细胞作为细胞生物学实验教学材料的优点和准备方法,可用其进行的细胞实验及具体用法等,旨在为细胞生物学实验教学中的选材提供参考。关键词小鼠腹水瘤细胞;细胞生物学实验;实验材料     

Binding and cytotoxicity of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and erythrocytes

The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of 125I-lectins to these cells gave biphasic lines except for HeLa cells at 0 degree C. The association constants of lectins for the three cell types at 37 degrees C were lower than those at 0 degree C. The numbers of total binding sites were estimated to be 7 to 16 X 10(7) per HeLa cell, 3 to 4 X 10(7) per Sarcoma 180 ascites tumor cell and 0.4 to 1 X 10(6) per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37 degrees C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1 M lactose, whereas no lectin was irreversibly bound at 0 degree C. In the case of erythrocytes, no lectin became irreversibly bound even at 37 degrees C. The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.     

灵芝多糖GLP 抑制小鼠骨肉瘤细胞S-180 在体内生长的作用机制

目的:探讨灵芝多糖成分(GLP)抑制肿瘤的作用机制。方法:在小鼠右腋皮下接种1×106TC-1细胞后7天后,用100mg/kg、200mg/kg和400mg/kg 3种剂量给小鼠口服灌胃给药20天,然后观察肿瘤的重量,并用ELISA检测小鼠血清中IL-2、IL-6和TNF-alpha,用流式细胞仪检测其外周血中CD4+和CD8+。结果:100mg/kg、200mg/kg和400mg/kg 3种剂量给小鼠口服灌胃给药20天,与对照组比较,抑瘤率分别可以达到53%、59%和58%,P<0.05;小鼠外周血血清中的IL-2从1.27ng/mL提高到了2.88ng/mL,P<0.05;TNF-α从1.05ng/mL提高到了1.82ng/mL,P<0.05;而IL-6则没有明显的变化。CD4+细胞水平升高(从54.80%提高到了58.27%),但差异无统计学意义(P>0.05);CD8+细胞明显增多(从24.15%提高到了45.36%),差异有统计学意义(P<0.05)。结论:GLP有明显抑瘤作用,但抑瘤作用与GLP剂量不存在依赖关系。GLP对肿瘤细胞生长的抑制是通过提高小鼠的细胞免疫能力来实现,而并非直接杀伤肿瘤细胞。     

Product inhibition studies of yeast phosphoglycerate kinase evaluating properties of multiple substrate binding sites.

Product inhibition studies on yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2- and two for 3-P-glycerate. 2. There is one catalytic centre per enzyme molecule. 3. The catalytic reaction primarily occurs at the 'first' or 'high affinity' MgATP2- and 3-P-glycerate binding sites. The 'second' set of sub-sites for these substrates are located in a region for regulation of the catalytic reaction. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. MgATP2- and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2- the apparent Ki value for 1,3-P2-glycerate increases from 3 microM to 20 microM.     

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.     

Macrovipera lebetina obtusa Snake Venom as a Modulator of Antitumor Effect in S-180 Sarcoma Mouse Model

Molecular Biology - Macrovipera lebetina obtusa (MLO) is a venomous snake endemic to Middle East. Here we describe the therapeutic potential of the MLO snake venom. In S-180 sarcoma-bearing mouse...     

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Abstract. The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1–24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5¢-amino-5¢-deoxythymidine (5¢-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.     

Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Two forms of deoxythymidine kinase from blast cells of acute myelocytic leukemia were identified by electrophoresis. One was associated mainly with the cytoplasm and the other with mitochondria. Both isozymes were separated and purified by differential affinity column chromatography which resulted in 2416- and 1634-fold purification of the cytoplasmic and mitochondrial enzymes, respectively. Affinity gel was prepared by linkage through position 3' of deoxythymidine. Each enzyme had the same electrophoretic mobility in the purified state as it did in the enzyme derived from the corresponding subcellular fraction of the homogenate. Thymidine phosphorylase was not retarded by the affinity column. The purified cytoplasmic and mitochondrial deoxythymidine kinase had different molecular weights, sensitivities to inhibition by ammonium sulfate, activation energies for the reaction and divalent cation requirements. Adenosine, guanosine, and cytosine 3':5'-monophosphates, putrescine, spermine, and spermidine were neither activators nor inhibitors of either deoxythymidine kinase.