scholarly journals Characterization of colchicine-binding activity in Dictyostelium discoideum

1978 ◽  
Vol 169 (3) ◽  
pp. 499-504 ◽  
Author(s):  
P Cappuccinelli ◽  
B D Hames
Keyword(s):  
Dictyostelium Discoideum ◽  
High Speed ◽  
Binding Protein ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Saturation Kinetics ◽  
Filter Assay ◽  
Kinetics Of ◽  
High Speed Supernatant

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.

scholarly journals Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect

1979 ◽  
Vol 184 (2) ◽  
pp. 431-440 ◽  
Author(s):  
G Mezzetti ◽  
R Loor ◽  
S Liao
Keyword(s):  
Binding Protein ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Ventral Prostate ◽  
Hormonal Effect ◽  
Rat Ventral Prostate ◽  
Acidic Fraction ◽  
Androgen Effect

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40–60% within 20–40 h after castration. This effect is reversed very rapidly within 15–30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.

Partial purification and characterization of a membrane-associated steroid-binding protein from Pseudomonas testosteroni

1982 ◽  
Vol 60 (8) ◽  
pp. 798-803 ◽  
Author(s):  
Mike Francis ◽  
Mamoru Watanabe
Keyword(s):  
Binding Protein ◽  
Membrane Vesicles ◽  
Activity Analysis ◽  
Binding Activity ◽  
Regulatory Function ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Steroid Binding ◽  
Steroid Binding Protein

A steroid-binding protein obtained from the supernatant of the final wash from the preparation of membrane vesicles was purified severalfold to near homogeneity. The protein binds C18 and C19 steroids but has the highest affinity for androstenedione (Kd = 1.6 × 10−10 M). The molecular weight is 51 000 – 58 000. Binding activity is slightly inhibited by Cu2+, Ca2+, and Mg2+ and completely inhibited by Zn2+. The protein has no detectable steroid degradative activity. Analysis of androstenedione binding revealed negative cooperativity of binding for this ligand and may indicate a regulatory function for this protein. It is postulated that this protein binds the steroid after testosterone is converted to androstenedione.

scholarly journals Novel prostaglandin dehydrogenase in rat skin

1983 ◽  
Vol 212 (1) ◽  
pp. 129-134 ◽  
Author(s):  
N Fincham ◽  
R Camp
Keyword(s):  
High Speed ◽  
Reaction Rates ◽  
Inhibitory Effects ◽  
Rat Skin ◽  
Prostaglandin F2 Alpha ◽  
Prostaglandin Dehydrogenase ◽  
The Mean ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
High Speed Supernatant

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.

Relationship of the cGMP-binding activity to the cGMP-specific phosphodiesterase in Dictyostelium discoideum

1986 ◽  
Vol 64 (6) ◽  
pp. 528-534 ◽  
Author(s):  
A. M. Parissenti ◽  
M. B. Coukell
Keyword(s):  
Dictyostelium Discoideum ◽  
Kinase Activity ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Wild Type ◽  
Dependent Protein ◽  
Low Levels ◽  
Relationship Of ◽  
The Relationship ◽  
Type Strains

Optimal conditions for assaying and stabilizing the soluble cGMP-binding activity in Dictyostelium discoideum were established. Using these procedures, we investigated the relationship between the cGMP-binding activity and the cGMP-specific phosphodiesterase in this organism. In wild-type strains, the binding and phosphodiesterase activities were found to be regulated differently during development. Also, stmF mutants, which possess very low levels of cGMP-specific phosphodiesterase activity, exhibited normal levels of cGMP-binding activity. Fractionation studies revealed that the binding and phosphodiesterase activities could be resolved by DEAE-cellulose chromatography. Finally, the effect of pH on cGMP binding was different from that reported for cGMP-mediated activation of the phosphodiesterase. Taken together, these results indicate that the cGMP-binding protein and the cGMP-specific phosphodiesterase are probably unrelated. In addition, the cGMP-binding activity is not associated with cGMP-stimulated kinase activity and it does not elute from DEAE-cellulose like the highly conserved cGMP-dependent protein kinases found in other systems.

scholarly journals Cloning and Characterization of a Gene,pbpF, Encoding a New Penicillin-Binding Protein, PBP2B, inStaphylococcus aureus

1999 ◽  
Vol 43 (7) ◽  
pp. 1578-1583 ◽  
Author(s):  
Hitoshi Komatsuzawa ◽  
Gil H. Choi ◽  
Kouji Ohta ◽  
Motoyuki Sugai ◽  
Monique T. Tran ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Protein Band ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Putative Signal Peptide ◽  
C Terminus ◽  
Histidine Tag

ABSTRACT A previously unrecognized penicillin binding protein (PBP) gene,pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed inEscherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region ofpbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

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