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1.
2.
Pressure-flow relationships in the isolated canine trachea 总被引:1,自引:0,他引:1
3.
Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells. 相似文献
4.
Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle 总被引:16,自引:3,他引:13
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Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle. 相似文献
5.
Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species. 总被引:1,自引:1,他引:0
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G B Knudson 《Applied microbiology》1985,49(4):975-980
Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light. 相似文献
6.
The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin. 相似文献
7.
Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal
structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure
independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316
and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire
a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet
circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary
structure of the fragments show cooperativity with a temperature of half-denaturationT
mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding
units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316
of thermolysin. 相似文献
8.
The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
M Emanuelli P Natalini N Raffaelli S Ruggieri A Vita G Magni 《Archives of biochemistry and biophysics》1992,298(1):29-34
Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity. 相似文献
10.
Vita Peri Boris Ajdukovic Paul Holland Balwant S. Tuana 《Molecular and cellular biochemistry》1994,130(1):57-65
Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys
Dystrophin
- T-tubule
Transverse tubule
- SDS-PAGE
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
- WGA
Wheat Germ Agglutinin
- Con A
Concanavalin A
- DHP
Dihydropyridine receptor
- FITC
Fluorescein Isothiocyanate Conjugate
- NAG
N-Acetyl-D-Glucosamine
- NP-40
NONIDET P-40
- PBS
Phosphate-Buffered Saline
- TBST
Tris Buffered Saline-Tween 相似文献