A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

To elucidate the role of protein conformation in the kinetics of adsorption at interfaces, seven structural intermediates of bovine serum albumin were prepared and their adsorption at the air/water interface was studied. Molecular area calculations indicated two distinct molecular processes, the first being the creation of an area, delta A1, for anchoring the molecule during the initial phase of adsorption and the second being the delta A2 cleared during subsequent reorientation and rearrangement of adsorbed molecules at the interface. The delta A1 values for all the albumin intermediates were the same, indicating that the initial work pi delta A1 needed to anchor the molecule at the interface was independent of solution conformation of the protein. Unlike delta A1, delta A2 exhibited a bell-shaped relationship with the extent of refolded state of the intermediates. Calculation of diffusion coefficients indicated that greater the unfolded state of the albumin intermediate, the greater was the diffusion coefficient. It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption. Specific, conformation-dependent, solute-solvent and solute-interface interactions also seem to influence the kinetics of adsorption of proteins.     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.     

互花米草、狐米草和大绳草茎秆的比较解剖观察

互花米草、狐米草和大绳草的表皮均由长细胞、短细胞(栓质细胞和硅质细胞)、盐腺和气孔器组成。它们成纵行交互排列。盐腺的结构与大米草相似,但三个种的盐腺和气孔器的数目不同,尤其以大绳草最多。它们的内部结构是由气道、不同大小的维管束、基本组织以及厚壁组织组成。然而,维管束的数目及厚壁组织的发育各不相同。狐米草和大绳草有高度木质化的厚壁组织细胞,而互花米草的厚壁组织木质化较弱。大绳草的维管束多于其他两种。     

Variations in ADP-ribosylation of nuclear scaffold proteins during the HeLa cell cycle

Cell cycle variations in ADP-ribosylation of nuclear scaffold proteins were determined. Nuclei of synchronized cells were isolated and labeled with [32P]NAD before nuclear scaffolds were obtained by digestion of DNA with DNase I and extraction of proteins with 2M NaCl. Autoradiograms revealed the three groups of "lamins" and a species identified as poly (ADP-ribose) polymerase to be the primary ADP-ribosylated proteins. The patterns of modification of nuclear scaffold proteins displayed similar features through the cell cycle. Radioactivity in the lamins increased from 20% in early-S phase to 40% in G1 phase of the next cell cycle.     

Cellulolytic Activity of Clostridium acetobutylicum

Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism.     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.