摘要:

AbstractThe membrane skeleton of the human erythrocyte consists of many short actin filaments that are multiply cross‐linked by long, flexible spectrin molecules into a continuous network in the plane of the membrane. The mechanical properties expected for this spectrin‐actin network can account for the tensile strength of the erythrocyte membrane and for the remarkable deformability of the cells, yet not for their characteristic biconcave shape. Recently, an authentic vertebrate myosin as well as a non‐muscle form of tropomyosin have been identified and purified from erythrocytes. The myosin is present with respect to the actin in an amount comparable to actin‐myosin ratios in other non‐muscle cells, and there is enough tropomyosin to almost completely coat all of the short actin filaments in the membrane skeleton. The implications of these unexpected discoveries for the molecular organization of the cytoskeleton are discussed, and a mechanism is proposed by which myosin could interact with the membrane‐associated actin filaments to influence erythrocyte shape and membrane

ISSN:0730-2312    

DOI:10.1002/jcb.240310102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe importance of the hydrophobic interaction in stabilizing native protein structure has long been appreciated. However, more than other component forces, this one has resisted quantitative description. We present two approximate methods of assessing the hydrophobic component to the free energy of protein folding. Both are expressed in terms of what can be calledhydrophobic momentsof the protein. The first method is intended to yield an approximate value for the hydrophobic energy. This energy is calculated from a set of atomic coordinates in terms of the hydrophobicity (or 0th hydrophobic moment) of each amino acid residue and its accessibility or lack of it to aqueous solvent. The second method considers the first moment of the hydrophobicity of a group of residues, the hydrophobic moment. Segments of secondary structure in folded proteins tend to have hydrophobic moments that oppose each other. For example, α‐helices on the protein surface tend to have one hydrophobic face and one hydrophilic face, with the hydrophilic face out towards the solvent. This pattern of organization is often apparent from a computer model of the protein that shows the magnitude and direction of the hydrophobic moment of each segment of secondary structure. Examples are given for the incorrectly folded structures of Novotný et al [J Mol Biol 177:787, 1984] and for the correct structures to which they corresp

ISSN:0730-2312    

DOI:10.1002/jcb.240310103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractBovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%–50% at pH 8.6 and 4 °C, and the half‐time for the refolding was approximately 60–75 min. We then refolded threonine‐neochymotrypsinogen, which is a two‐chain structure held together by disulfide bonds and produced on cleavage of Tyr 146‐Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866–9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino‐ and carboxyl‐terminal domains, were separated on Sephadex G‐75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1 : 5 to 5 : 1 were refolded with yields of 21–28%. The lack of dependence on the concentration of either fragmènt and the relatively high yields suggest independent folding of the amino‐ and carboxyl‐terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper

ISSN:0730-2312    

DOI:10.1002/jcb.240310104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractA rabbit antiserum (A2) directed against the detergent‐solubilized fraction of the simian virus 40‐transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two‐dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2–6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two‐dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6–7.2) and an acidic (6.4–6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12–16‐day mouse embryos show the immunoprecipitated set of polypeptides only in the 16‐day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic, cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18‐day‐old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12‐day‐old mice even when the cultures were propagated at least‐15 times. This set of polypeptides is also present in simian virus 40‐transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40‐transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus‐transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in meta

ISSN:0730-2312    

DOI:10.1002/jcb.240310105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractWe have recently described the isolation and purification to homogeneity of a new Sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the Sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6–8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequenc

ISSN:0730-2312    

DOI:10.1002/jcb.240310106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractWe have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single‐stranded DNA and pulse‐labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited.When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed.Preparations of purified, high‐molecular weight, double‐stranded DNA contain variable amounts of fast‐sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation.These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts.Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average, length practically all DNA was released. The last 1–4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3in metrizamide density gradients and showing less than 4% retention on filters.At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA.It was shown, however, that digestion of nuclear lamina‐DNA complex with EcoRI or Hae III led to the formation of DNA‐protein aggregates, which banded at 1.35 g/cm3in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.These results argue against the existence of direct tight bonds between DNA and nuclear lamina in vivo but demonstrate that such bonds can be generated under certain cond

ISSN:0730-2312    

DOI:10.1002/jcb.240310107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy‐dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte–leukoagglutinin (LPHA) system, which closely mimics the IM9‐insulin model. Reliable estimates of total binding capacity (728 ng/106cells) essential to our report were calculated from a wide database by the least‐squares model. At occupancies ≥ 0.085, receptors are associated with low and fixed affinity (1.5 × 106M−1), whereas at occupancies ⩽ 0.085, affinity is high and fixed (1.8 × 108M−1) or high but variable (1 × 107M−1to 1.5 × 106M−1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor–ligand complex dissociation velocity over a wide range of occupancies (0.01–0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of ≅ 0.004 and is magnified as ligand binding to high affinity receptors increases up to ≅ 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulatin

ISSN:0730-2312    

DOI:10.1002/jcb.240310108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240310101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY