Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation.

p60src, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the alpha-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotide-directed mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p60src with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p60src, the inability of this mutant p60src protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.     

Molecular weight of bacteriorhodopsin solubilized in Triton X-100.

Bacteriorhodopsin from Halobacterium halobium has been solubilized in the nonionic detergent Triton X-100. The circular dichroic spectrum and hydrodynamic properties indicate that the structure of this protein in the detergent is not significantly altered from that of the native membrane-bound form. Bacteriorhodopsin is monomeric under the conditions of solubilization with a molecular weight of 24,250+/-2,000 and binds approximately one micelle of Triton X-100.     

Bovine papillomavirus E7 transformation function correlates with cellular p600 protein binding

The E7 oncoprotein of bovine papillomavirus type 1 (BPV-1) is required for the full transformation activity of the virus. However, the mechanism by which E7 contributes to cellular transformation is unknown. To address this question, we used the proteomic approach of tandem affinity purification to identify cellular proteins that are in complex with E7, and identified the 600-kDa protein, p600, as a binding partner of E7. The ability of E7 to complex with p600 correlated with its ability to enhance anchorage independence of BPV-1 E6-expressing cells. Furthermore, E7 mutant proteins impaired in their ability to bind p600 were transformation defective. Additionally, knockdown of p600 reduced transformation of cells expressing both BPV-1 E6 and E7, as well as E6 alone, suggesting that the ability of E7 to transformed cells is mediated, at least in part, through its ability to bind p600. These data complement work that shows that HPV16 E7 also interacts with p600, and that this interaction correlates with the ability of HPV16 E7 to transform cells. These studies thus identify p600 as a shared target of the E7 proteins of multiple papillomaviruses.     

Long-wavelength-absorbing forms of bacteriochlorophyll a in solutions of Triton X-100

At least three forms of Triton X-100-solubilized bacteriochlorophyll a (BChl a) have been characterized by UV/visible/near-IR absorption and CD spectra. One, absorbing at 770 nm, is similar to a monomeric solution in methanol. The two others have strongly red-shifted absorption peaks (860 nm and 930, 835 nm) and intense and complex CD bands in this region, indicative of strong interaction of at least two and three molecules of BChl a, respectively.     

Triton X-100浸出抗原ELISA检测牛结核

用Triton X-100自牛分枝杆菌(M.bovis)浸出物为抗原,建立了ELISA检测牛结核血清抗体的方法,阴性平均OD值(?)为0.09,标准差(SD)为0.10,以(?)+3SD为临界值检测了结核菌素(OT)变态反应阳性牛118头,ELISA阳性率为60％;检测健康牛的阴性符合率为97.1％,假阳性率2.9％,与其它疾病的交叉反应为1.75％。ELISA与病变及细菌培养有较高的符合率。     

曲拉通X-100对制备脱细胞真皮基质影响的实验研究

目的探讨由曲拉通X-100制备脱细胞真皮基质(ADM)的最佳浓度及作用时间,为表皮细胞种植于ADM形成组织工程化口腔黏膜提供实验依据。方法取大鼠2cm×2cm大小的皮肤56块,置于0.25% Dispase试剂中4C下孵育48小时,轻轻去掉表皮,随机分成7组,分别浸入0.1%、0.2%、0.3%、0.5%、1%、3%、10%的曲拉通X-100中作用36～80小时。取标本制作组织切片,HE染色,大体、光镜观察。结果光镜检查结果显示,制备出的脱细胞真皮基质主要由胶原纤维网架构成;曲拉通X-100浓度较低(0.1%～0.5%)时,皮肤脱净细胞成分的时间随浓度升高而逐渐缩短,各浓度间有明显差异(P均<0.01),而当浓度>0.5%时,皮肤脱净细胞时间与浓度无关,组间无显著差异(P>0.05)。结论曲拉通X-100制备大鼠ADM的最佳浓度为0.5%,时间为(45±2.82)小时。     

Characterization of avian and viral p60src proteins expressed in yeast.

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.     

Fine structural mapping of a critical NH2-terminal region of p60src.

We have recently demonstrated that an NH2-terminal sequence required for myristylation and membrane association of the Rous sarcoma virus transforming protein, p60src, is contained within amino acids 2-14 [Cross, F.R., Garber, E. A., Pellman, D. & Hanafusa, H. (1984) Mol. Cell. Biol. 4, 1834-1842]. This sequence is also required for cell transformation. We have now constructed five mutants of Rous sarcoma virus that contain alterations in the src sequence coding for these 14 amino acids. Mutants encoding src proteins with a peptide insertion between amino acids 1 and 2, or peptide substitutions for amino acids 2-4, 3-4, or 7-15, were transformation-defective. The src proteins of these mutants differed from the wild-type protein in that they were not myristylated and did not fractionate with the plasma membrane of infected cells. The fifth mutant encoded a src protein with a short peptide substituted for amino acids 11-15. This protein was myristylated and plasma membrane associated, and the virus transformed cells. We therefore conclude that a sequence required for myristylation and membrane association of p60src is located within the first 7-10 amino acids of the src protein, and that p60src myristylation and membrane association are required for cell transformation. Consistent with this idea, we have isolated four transforming revertants from one of the transformation-defective mutants. The src proteins of all four revertants were found to be myristylated and membrane associated.     

Anomalous interaction of the acetylcholine receptor protein with the nonionic detergent Triton X-114.

Integral membrane proteins that form water-filled channels through membranes often exist as aggregates of similar or identical subunits spanning the membrane. It has been suggested that the insertion into the membrane of the channel-forming domains of the subunits may impart unusual structural features to the membrane-intercalated portions of the protein. To test this proposal, we have investigated the interaction of a multisubunit channel-forming integral membrane protein, the acetylcholine receptor protein, with the nonionic detergent Triton X-114. Whereas non-channel-forming integral membrane proteins that have heretofore been studied form mixed micelles with the detergent, the acetylcholine receptor was excluded from the Triton X-114 micelles. The structural implications of this result are discussed.     

Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM)

Changes in HeLa cell morphology, membrane permeability, and viability caused by the presence of Triton X-100 (TX100), a nonionic surfactant, were studied by scanning electrochemical microscopy (SECM). No change in membrane permeability was found at concentrations of 0.15 mM or lower during an experimental period of 30 to 60 min. Permeability of the cell membrane to the otherwise impermeable, highly charged hydrophilic molecule ferrocyanide was seen starting at concentrations of TX100 of about 0.17 mM. This concentration level of TX100 did not affect cell viability. Based on a simulation model, the membrane permeability for ferrocyanide molecules passing though the live cell membrane was 6.5 ± 2.0 × 10^-6 m/s. Cells underwent irreversible permeabilization of the membrane and structural collapse when the TX100 concentration reached the critical micelle concentration (CMC), in the range of 0.19 to 0.20 mM. The impermeability of ferrocyanide molecules in the absence of surfactant was also used to determine the height and diameter of a single living cell with the aid of the approach curve and probe scan methods in SECM.     

Cortical benzodiazepine receptor binding in a rabbit model of hepatic encephalopathy: The effect of Triton X-100 on receptor solubilization

Increased benzodiazepine (BZ) receptor density has been reported in brains of rabbits with hepatic encephalopathy (HE) due to galactosamine (GalN)-induced fulminant hepatic failure (FHF). These data were generated using detergent-Triton X-100-treated neural membranes. While performing further studies it was noted that the increase in BZ receptor density was not demonstrable when Triton X-100 preparation was not employed. Accordingly the binding of [³H] flunitrazepam, a BZ ligand, to neural membranes from cortices of normal rabbits and rabbits with HE due to (GalN)-induced FHF was studied with and without detergent preparation. Scatchard plot analysis of the binding data indicated that when no detergent was employed, the apparent affinity and density of BZ receptors were similar for control membranes and membranes from animals in HE. BZ receptors from animals in HE were shown to be more resistant to solubilization by Triton than control membranes. These findings (a) afford a potential explanation for the apparent increase in density of BZ receptors in this model when Triton treatment of neural membranes is utilized and (b) suggest that recent evidence for increased GABAergic tone in the syndrome of HE is not dependent on an increased density of BZ receptors.     

Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli.

We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected. Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells. Partially purified src protein from E. coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells.     

Synthetic peptide fragment of src gene product inhibits the src protein kinase and crossreacts immunologically with avian onc kinases and cellular phosphoproteins.

All the known avian sarcoma viruses have associated protein kinase activities that phosphorylate tyrosine residues of their target proteins. A decapeptide fragment of pp60src of Rous sarcoma virus (RSV), residues 415-424, and an analog of that sequence have been chemically synthesized by solid-phase methods. The two decapeptides were not phosphorylated by pp60src of RSV, P90 of Y73 avian sarcoma virus, or P140 of Fujinami sarcoma virus. However, both peptides were able to inhibit competitively the kinase activities associated with the transforming proteins. Antiserum was raised against one of the peptides and IgG was purified from the serum by affinity chromatography. The antibody was able to precipitate pp60src of RSV as well as P90 of Y73 virus from cells infected with these viruses. The antibody also precipitated a number of high molecular weight phosphoproteins from normal chicken and rat fibroblasts and from several lines of virus-transformed cells.     

Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.

Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.     

NH2-terminal sequences of two src proteins that cause aberrant transformation.

Two isolates of recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, transform cells in culture, but have greatly reduced in vivo tumorigenicity. The src proteins of rASV157 and rASV1702 have alterations within their NH2 termini, are not myristoylated, and have an altered subcellular localization. We have molecularly cloned and determined the nucleotide sequences of the src genes of rASV157 and rASV1702. We found that their src proteins have unusual NH2 termini: the rASV157 src protein NH2 terminus consists of 30 amino acids of the env signal peptide attached to Ser-6 of the src sequence, while the rASV1702 src protein NH2 terminus consists of 45 amino acids of the env signal peptide attached to Ala-76 of the src sequence. Expression of recombinant Rous sarcoma virus constructs containing the molecularly cloned rASV src genes produced src proteins with the same properties as those of the parental viruses. Our results suggest that the NH2-terminal structures are responsible for many unusual properties of the mutant src proteins.     

The temperature dependence of the calcium paradox: Enzymatic,functional and morphological correlates of cellular injury

An isolated rat heart preparation was used to characterize the temperature dependence of the calcium paradox and also to assess the validity of various indices of hypothermic protection. Hearts were subjected to 10-min periods of calcium depletion at various degrees of hypothermia followed by 20 min of normothermic calcium repletion. Using enzyme or protein leakage during calcium repletion as an index of hypothermic protection during calcium depletion, paradox injury was reduced extensively by relatively moderate hypothermia. Thus, depletion at 29°C reduced total creatine kinase leakage by 57 ± 4% from 1585 ± 24 IU/g dry wt to 677 ± 63 IU/g dry wt and at 25°C leakage was reduced by 85 ± 4% from 1585 ± 24 IU/g dry wt to 237 ± 71 IU/g wt. However, upon calcium repletion there was no recovery of contractile function. It was not until the myocardial depletion temperature was reduced to 20°C that some functional recovery occurred. Under these circumstances cumulative creatine kinase leakage was reduced to below 88 IU/g dry wt, 6% of its normothermic value and protein leakage was undetectable. Functional recovery was not complete until the temperature was reduced to 15°C or below. Correlation of cumulative enzyme leakage with functional recovery suggested a narrow release threshold (50 to 100 IU/g dry wt) above which no recovery occurred and below which a full recovery could be confidently predicted. Morphological assessments indicated an all-or-none phenomenon; thus although increasingly severe hypothermia progressively reduced the percent of cells that sustained damage (as opposed to the degree of damage in all cells), it was not until 100% of cells appeared ultrastructurally undamaged that functional recovery was observed. Calcium-free perfusion at 4°C protected the intercalated discs from gross lesions and prevented the separation of the external lamina from the surface coat. Our results also stress the heterogeneity of tissue injury and hypothermic protection and in addition shed further light upon the component mechanisms contributing to calcium injury.     

Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.     

Biochemical and morphological correlates of cardiac ischaemia: Contractile proteins.

Experimentally-induced ischaemia in the dog heart was produced by ligating the left circumflex artery. Myosin B isolated from the ischaemic portion of the myocardium differed from myosin B isolated from control tissue in its diminished response to the calcium chelator ethyleneglycol bis (beta-amino-ethylether)-N, N'-tetraacetic acid (EGTA). In the presence of EGTA, ischaemic myosin B required 2.5 +/- 0.5 min for completion of superprecipitation, whereas control myosin B required 6.6 +/- 2.5 min. Likewise, the Mg++ -activated ATPase activity of ischaemic myosin B was inhibited by EGTA to a lesser degree than control myosin B. Experiments with reconstituted myosin B using desensitized control myosin B and regulatory proteins suggest that ischaemia induces changes in the regulatory proteins (troponin and tropomyosin).