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1.
Summary Mannose is incorporated in monkey liver chromatin by the means of a nuclear membrane mannosyl-transferase.14C-labelled chromatin is dissociated either by sulfuric acid or 6 M urea and 0.4 M GuCl. The fractions then enriched in non-histone14C-labelled proteins are excluded from Ultro-gel AcA 202, their analysis in SDS-polyacrylamide gel electrophoresis shows that radioactivity fits with one major protein band, confirming the presence of at least a non-histone protein labelled with mannose in monkey liver chromatin, with an apparent molecular weight of 13 000. 相似文献
2.
Hikaru Hemmi Takashi Kumazaki Shuichi Kojima Takuya Yoshida Tadayasu Ohkubo Hideyoshi Yokosawa Yuji Kobayashi 《Journal of peptide science》2011,17(8):595-600
P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys14? Cys39 bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys14? Cys39 bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant (Khyd°) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in Khyd°, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
3.
Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells 总被引:2,自引:0,他引:2
R D Platz G S Stein L J Kleinsmith 《Biochemical and biophysical research communications》1973,51(3):735-740
The phosphorylation of non-histone chromatin proteins in synchronized HeLa S3 cells was studied in 5 phases of the cell cycle: mitosis, G1, early and late S, and G2. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G1 and G2, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with 32P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle. 相似文献
4.
T. Kobayashi Y. Hakamada J. Hitomi K. Koike S. Ito 《Applied microbiology and biotechnology》1996,45(1-2):63-71
Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease,
designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two
minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease
had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein
was observed at pH 11.0 and at 55°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE,
although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and
at 60°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease.
The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values
and at 5°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease
at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.
Received: 12 December 1994/Received last revision: 9 June 1995/Accepted: 31 July 1995 相似文献
5.
The distribution of nuclear non-histone proteins and their labelling with [14C]amino acids or [32P]O4 in rat ventral prostate cells undergoing hypertrophy (cell growth) or hyperplasia (cell division) were compared by SDS polyacrylamide gel electrophoresis. Marked quantitative changes in these proteins accompanied castration and replacement of hormone, but no absolute qualitative differences in their staining patterns were established. Preparations from cells undergoing hyperplasia were augmented in high molecular weight proteins and contained fewer proteins of <20000 D. The increased amounts of nuclear non-histone proteins from hormone-treated castrated rats were highly labelled by [14C]amino acids, but the molecular weights of radioactive proteins from cells undergoing hypertrophy were less diverse than those from replicating cells, Phosphorylation of nuclear non-histone proteins from short-term and long-term castrates, treated with testosterone propionate, was 170 and 60% greater than their controls. Proteins from 20–45 × 103 D were actively phosphorylated. Nuclear extracts from dividing cells contained additional radioactive high molecular weight proteins and fewer phosphorylated lower molecular weight components. The distribution of phosphorylated proteins and newly synthesized proteins was dissimilar. Quantitative and possible qualitative differences in staining of nuclear proteins isolated in N-ethylmalleamideurea-phosphate buffer from normal or hormone-treated castrated rats were accentuated when they were separated by charge at pH 2.8. In replicating cells, a more generalized synthesis of acidic nuclear proteins from all molecular weight classes occurred, which were not as highly phosphorylated as less heterogeneous nuclear proteins from cells undergoing hypertrophy. Examination of the immediate and the subsequent events following androgen-induced cellular hypertrophy or hyperplasia in the ventral prostate permits their comparison in the same tissue. 相似文献
6.
An enzymatic assay is described which quantitates radiolabeled type IV basement membrane collagen in the presence of large amounts of other proteins. A partially purified neutral protease is used which cleaves type IV collagen into fragments at 37°C which are not precipitated at 1.3% (final concentration) trichloroacetic acid-tannic acid. The kinetics of type IV collagen digestion by this enzyme are not significantly altered by the presence of a 10-fold excess of type III collagen. [14C]Tryptophan-labeled control proteins prepared from fibroblast cultures are not degraded significantly by this protease in the presence of 2.5 mmN-ethylmaleimide. The proportion of type IV collagen in a mixture of labeled placenta collagenous proteins was calculated after separate digestions with the type IV collagenolytic activity and bacterial collagenase: this value compared favorably with the proportion of type IV collagen estimated by gel electrophoresis. 相似文献
7.
Chintaman G. Sahasrabuddhe Ramesh C. Adlakha Potu N. Rao 《Experimental cell research》1984,153(2):439-450
Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis. 相似文献
8.
Shun-ichiro Kubota Koichi Suzuki Kazutomo Imahori 《Biochemical and biophysical research communications》1981,100(3):1189-1194
A Ca2+--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca2+ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained. 相似文献
9.
Stephen F. Cleary Guanghui Cao Li-Ming Liu Patsy M. Egle Keith R. Shelton 《Bioelectromagnetics》1997,18(7):499-505
The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [35S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd++ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc. 相似文献
10.
α-Amylase was found to be the main protein secreted byBacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45°C, respectively. A change in temperature
can affect the pattern of proteins secreted as detected by gel electrophoresis.14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30°C
than that at 40 or 45°C. The effect of temperature on α-amylase synthesis as determined by its enzymic activity and on the
extracellular protein synthesis followed a similar pattern. 相似文献
11.
Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells 总被引:3,自引:0,他引:3
P Adamietz K Klapproth H Hilz 《Biochemical and biophysical research communications》1979,91(4):1232-1238
The (ADP-ribose)n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (3H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADP-ribose)n residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (3H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (Mr = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors. 相似文献
12.
Tubulin in high-speed supernatants of brain undergoes an alternate form of polymerization into structures that resemble membranes rather than microtubules. The reaction required elevated temperature (37°C) and was prevented by 1mM CaCl2 or 10?4 M maytansine. The membraneous material was composed of tubulin (80%) and microtubule-associated proteins (8%) and contained phospholipids. The tubulin was identified on the basis of comigration in two-dimensional gel electrophoresis and colchicine-binding activity. 相似文献
13.
BISWENDU B. GOSWAMI SYAMALIMA DUBE PRABIR BHATTACHARYYA SANDIP N. SINHA BIMAL K. RAY D. K. DUBE S. C. ROY 《Physiologia plantarum》1975,35(2):111-114
Proteins synthesized in vitro by mitochondria isolated from 48-h germinating seeds of Vigna sinensis (L.) Savi and incubated in the presence of 14C-labelled amino acids from Chlorella protein hydrolysate, have been found associated with nine products separable by acrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Cytoplasmic contribution to these products was practically eliminated by the use of cycloheximide. Most of the radioactivity was incorporated into proteins having molecular weights between 10,000 and 65,000 as determined by comparing their electrophoretic mobilities with those of standard, reference proteins. 相似文献
14.
Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about
1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold
purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified
enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active
at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C
up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up
to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating
the potential role of this enzyme for detergent application. 相似文献
15.
The latent coupling factor (F1)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F1 from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits α, β, γ, δ, and ? and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with 14C-labelled, F1-ATPase prepared from cells grown on medium containing [U-14C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3. 相似文献
16.
Calf brain synaptic plasma membranes were saturated under extracellular conditions with [14C]glutamic acid and the resulting labelled membranes fractionated with 0.9% NaCl, distilled water,n-butanol-water, 0.05 mol/L NaOH and 0.5% Triton X-100 solutions in this order. Triton X-100 was the most effective solubilizer, liberating altogether about 24% of the membrane proteins, but only 4–7% of the label, while NaOH was the most potent solubilizer for the protein-bound label (50–70%). Slab gel electrophoresis showed significant qualitative differences in the banding patterns of the protein extracts, but only two fractions, a low-molecular weight (around 15 kd) and a high-molecular weight (>200 kd) fraction bound [14C]glutamate. 相似文献
17.
Jana Brabcová Jiří Kindl Irena Valterová Iva Pichová Marie Zarevúcka Jana Brabcová Michal Jágr Ivan Mikšík 《Archives of insect biochemistry and physiology》2013,82(3):117-128
A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N‐succinyl‐L‐alanyl‐L‐alanyl‐L‐prolyl‐L‐phenyl‐alanine 4‐nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55 ± 0.042 mM and 0.714 ± 0.056 μmol p‐nitroalanine produced min?1 mg protein?1, respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N‐tosyl‐L‐phenylalanine chloromethyl ketone, which indicated the trypsin‐like but not chymotrypsin‐like specificity. The identity of the serine protease was confirmed by nanoliquid‐tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence. 相似文献
18.
Ian D. Reid 《Biotechnology Techniques》1991,5(3):215-218
Summary Molecular weight distributions of 14C-labelled alkali-soluble lignins and their biodegradation products can be quickly and conveniently determined by gel filtration on a Pharmacia Superose 12 column. Elution with a 1:1 mixture of ethanol and 0.1 M NaOH prevents adsorption of the lignins on the gel. 相似文献
19.
Pea membranes were incubated with UDP-[14C]galactose and sequentially extracted with lipid solvents and 2% sodium dodecyl sulfate (SDS). At least three-quarters of the products were SDS-soluble. All fractions contained some [14C]glucose, indicating the presence of an active epimerase which, however, could be inhibited by ADP-ribose. The chloroform-methanol extract contained mainly neutral galactosyl lipids and a small amount of dolicyl monophosphoryl glucose. The chloroform-methanol-water extract contained trace amounts of lipid-linked galactosyl oligosaccharide with properties comparable to polyisoprenyl pyrophosphoryl derivatives. Polyacrylamide gel electrophoresis of SDS-soluble products indicated the formation of both immobile and mobile components with similar size distribution (Sepharose CL-6B). The mobile component only was susceptible to hydrolysis by protease. Periodate oxidation analysis of SDS-soluble and -insoluble products indicated that they were composed mainly of 1 → 6 galactosyl residues, i.e. as in many arabinogalactan proteins and arabinogalactans. 相似文献
20.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42 总被引:1,自引:0,他引:1
Kazan D Denizci AA Oner MN Erarslan A 《Journal of industrial microbiology & biotechnology》2005,32(8):335-344
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease
activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate
by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum
temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal
pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses
of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis
by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h
at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was
not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively. 相似文献