Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.     

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.     

Stability of proteins in guanidine·HCl solutions

A new method has been described for the study of the denaturation of proteins by guanidine·HCl. The pH of an unbuffered solution of protein is monitored as Gu·HCl solution at a constant rate is run into the protein solution. As the protein undergoes the transition from the native to denatured state a significant and fairly abrupt change in the pH is observed. The behaviour of 14 proteins has been explored. Lysozyme, pepsin, α-lactalbumin, lima bean trypsin inhibtor, and insulin failed to yield satisfactory transitions by this method. The method is entirely satisfactory with the other proteins. The maximum stabilities of the proteins in respect to Gu·HCl concentration and pH, and at zero rate (by extrapolation) of delivery of Gu·HCl have been studied. The relative stabilities of the proteins differ greatly. The stabilities are not related in a simple way to any known parameter. To supplement the pH change method outlined, the viscosities of nine proteins have been studied as a function of Gu·HCl concentration. A useful empirical equation relating the viscosity of a Gu·HCl solution to its molar concentration has been developed. The linear relationship between the relative fluidity and protein concentration has been confirmed and the intrinsic viscosities of the proteins have been calculated. The viscosities of the proteins vary significantly in respect to Gu·HCl concentration. All of the proteins undergo transitions from the native to the denatured state as shown by increases in the viscosities of their solutions. These transitions coincide closely with those found by the pH change method in respect to Gu·HCl concentration and pH. After the initial transition, the proteins continue to expand (viscosity increases) as the Gu·HCl concentration increases, indicating that the denatured state consists of many molecular configurations likely differing little in their energy content. The presence of disulfide bonds tends to limit the expansion of the protein molecules.     

Antigenic and morphologic comparisons of Ibaraki and bluetongue viruses.

Bluetongue disease virus, type 10, and Ibaraki disease virus, which causes a bluetongue-like disease of cattle, were compared to determine whether they are the same or different viruses. They were similar in morphology, but neutralization tests, complement-fixation tests, and ferritin tagging indicated that they have antigenic differences. Therefore, they should be considered as different viruses. Two other viruses of this group, African horsesickness and equine encephalosis, were included to show that Ibaraki and bluetongue had developmental morphological features that could be used to differentiate them from the two equine viruses.     

Temperature dependence of partial volumes of proteins

The change of the apparent partial specific volumes of egg albumin, bovine serum albumin, bovine methemoglobin, β-lactoglobulin, and lysozyme with temperature through the thermal transitions of the proteins have been measured with dilatometers. Four regions in the plot of the apparent partial specific volumes against temperature can be recognized: (1) linear sections extending from 25°C up to 45–50°C: (2) a decrease in slope between 50°C and 60°C; (3) a sharp increase in slope with increasing temperature coinciding with the appearance of heat coagulation of the protein and followed by (4) a decrease in the slope. The return of the protein samples to 25°C yields linear relations between the apparent partial specific volumes of the heat-denatured proteins and the decreasing temperature.