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31.
The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent Mr of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the Mr 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the Mr 17,700 polypeptide and of 42 residues for the Mr 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI
photosystem I
- P700
reaction center of PSI
- SDS
sodium dodecylsulfate
- TBS
tris-buffered saline
- TTBS
TBS containing Tween-20 相似文献
32.
P. M. Abuja M. Schmuck I. Pilz P. Tomme M. Claeyssens H. Esterbauer 《European biophysics journal : EBJ》1988,15(6):339-342
Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, Dmax=6.5 nm) and for the intact enzyme (Rg=4.27 nm, Dmax=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS
small angle X-ray scattering
- SDS-PAGE
SDS-polyacrylamide gel electrophoresis
- IEF-PAG
polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91))
- Dmax
maximum diameter
- Rg
radius of gyration 相似文献
33.
Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated. 相似文献
34.
P L Black H Phillips H R Tribble R Pennington M Schneider J E Talmadge 《Journal of cellular biochemistry》1988,36(4):377-392
The mechanism of therapeutic activity of recombinant murine interferon-gamma (rMu IFN-gamma) and the IFN inducer polyinosinic-polycytidylic acid solubilized with poly-L-lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN-gamma or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN-gamma and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN-gamma and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN-gamma, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN-gamma. LAK activity at any site did not correlate with the therapeutic activity of either agent. 相似文献
35.
测定了3T3细胞、人和大鼠一些组织中DNA拓扑异构酶Ⅰ的活性;估计了核酸内切酶对拓扑酶Ⅰ松弛活性测定的干扰程度;发现增殖组织全细胞抽提液中酶比活高于正常分化组织,而且在异常增殖组织中酶比活的增高更为显著。 相似文献
36.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
37.
Allison A. Welder Tina Machu Steven W. Leslie Richard E. Wilcox June Bradlaw Daniel Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):771-777
Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models
for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding,
beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained
from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude
membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B
max
(42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K
D
of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after
3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7
M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess
saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable
than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology
Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration. 相似文献
38.
Kenneth P. Karey Terry L. Riss B. Daniel Burleigh David Parker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1107-1113
Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized,
resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (125I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation
at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding
sites with K
d
=59.6 pM and an estimated 1.57×105 receptors/cell. Half-maximal displacement of bound125I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective
competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal
growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for125I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more
tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that125I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
39.
David K. Grandy Richard Leduc Haripriya Makam Thomas Flanagan Emanuel J. Diliberto Jr. Olivier Civelli O. Humberto Viveros 《Cellular and molecular neurobiology》1992,12(2):185-192
1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain. 相似文献
40.
Shiaw-Der Yang Jen-Shin Song Yao-Tsung Hsieh Hui-Wen Liu Wen-Hsiung Chan 《Journal of Protein Chemistry》1992,11(5):539-546
The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca2+-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK
m
value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission. 相似文献