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1.
Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism 总被引:7,自引:0,他引:7
Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM
Crassulacean acid metabolism
- DTT
dithiothreitol
- kDa
kilodalton
- PAGE
polyacrylamide gel electrophoresis
- PPiase
pyrophosphatase
- SEC
size exclusion chromatography
- SDS
sodium dodecyl sulfate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
2.
Phosphoglycolate phosphatase (EC 3.1.3.18), isolated from maize leaf bundle sheath, was purified 200-fold to a specific activity of about 99 mol mg-1 protein · min-1. The purification procedure included Sephadex G-75 filtration, and diethylaminoethyl-cellulose and Phospho-Ultrogel A6R chromatography. This partially purified enzyme exhibited optimum activity over a broad pH range, from pH 6.3 to pH 8.0. It displayed a very high degree of specificity for phosphoglycolate and required a divalent cation to be active; Mg2+ was the most effective activator. Saturation curves of the Michaelis-Menten type were observed both with phosphoglycolate (Km=0.57 mmol·l-1) and with Mg2+ (Km=0.015 mmol·l-1). The activation constant for Mg2+ was unchanged when the pH was raised from 7.0 to 8.0. These results indicate that variations of stromal pH and Mg2+ during the darklight transition could not directly modifity the activity of the phosphoglycolate phosphatase in maize bundle-sheath chloroplasts. The undissociated protein showed a pI of 4.95, as determined by isoelectric focusing. For the native phosphatase a molecular mass of about 61 500 Da was estimated by polyacrylamide gradient gel electrophoresis. The subunit was found to have a relative molecular mass of 31 500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is concluded that maize phosphoglycolate phosphatase is a dimer.Abbreviations DEAE
diethylaminoethyl
- P-glycolate phosphatase
phosphoglycolate phosphatase
- P-glycolate
phosphoglycolate
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl[glycine
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
3.
A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA53 to GA44 was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe2+ stimulated the reaction, but externally added Fe2+ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe2+ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA53 was converted to both GA44 and GA19. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA
bovine serum albumin
- DEAE
diethylaminoethyl
- DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- GAn
gibberellin An
- HPLC
high-performance liquid chromatography
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
4.
Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl2 was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a 44 structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH
acetaldehyde dehydrogenase
- ADH
alcohol dehydrogenase
- CHES
2-(N-cyclohexylamino)-ethanesulfonate
- DTE
dithioerythritol
- KP-buffer
25 mM K-PO4, pH 7.5, containing, 4 mM DTE
- MES
2-(N-morpholino)-ethanesulfonate
- TAPS
N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate 相似文献
5.
Evidence is presented that the organelles of Lemna minor do not degrade as functional units. The proteins of Lemna show wide differences in their rates of degradation and ribulose bisphosphate carboxylase (EC 4.1.1.39) has a particularly slow rate of degradation. Contrary to some earlier evidence, we found no correlation between the rate of soluble-protein degradation and either charge or size of proteins. We could find no correlation between protein degradation and subunit size in any of the organelles of Lemna.Abbreviations FPLC
fast protein liquid chromatography
- PAGE
polyacrylamide gel electrophoresis
- RuBPCase
ribulose-1,5-bisphosphate carboxylase
- SDS
sodium dodecylsulphate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
6.
Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [35S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS
citrate synthase
- EDTA
ethylenediaminetetraacetic acid,-acetate
- GAPDH
NADP+-glyceraldehyde-3-phosphate dehydrogenase
- rRNA
ribosomal RNA
- SDS
sodium dodecyl sulphate
- SDS-PAGE
SDS-polyacrylamide gel electrophoresis
- Tris
2-amino-2(hydroxymethyl)-1,3-propanediol 相似文献
7.
Sequestered particles of phytochrome (SAPs) were partially purified from red-light-irradiated oat coleoptiles. Phytochrome pelletability was enhanced by using buffers containing 10 mM Mg2+ or high concentrations (0.6–0.8 M) of orthophosphate (Pi). Combining the pelletability of phytochrome in the presence of Mg2+ with that in the presence of 0.6 Pi resulted in a strong enrichment (about 100-fold) of pelletable phytochrome. Antisera were raised against Mg2+-Pi-pellets from darkgrown seedlings. Using these antisera, no evidence was found by Western blotting and immunocytochemistry that SAPs contain major proteins other than phytochrome. The major contamination of these enriched SAP preparations consisted of protein crystals which are probably catalase. The preparations contained methyltransferase and protein-kinase activities which were not associated with SAPs. Phytochrome purified from SAPs served as a substrate for protein-kinase activity but not for the methyltransferase activity. Phytochrome itself did not show any kinase activity.Abbreviations ME
2-mercaptoethanol
- PAGE
polyacrylamide gel electrophoresis
- Pfr
far-red-light-absorbing form of phytochrome
- PMSF
phenylmethylsulfonyl fluoride
- SAP
sequestered area of phytochrome
- SDS
sodium dodecyl sulfate
This work was supported by Deutsche Forschungsgemeinschaft. The competent technical assistance of Karin Fischer is gratefully acknowledged. 相似文献
8.
Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin 总被引:1,自引:0,他引:1
The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[3H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[3H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([3H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [3H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC
[(4-azidobenzoyl)-ethylenediamine]-fusicoccin
- ABE-NHS
(4-azidobenzoyl)-N-hydroxysuccinimide ester
- FC
fusicoccin
- FCBP
fusicoccin-binding protein
- FCol
9-norfusicoccin-8-alcohol
- MAB
monoclonal antibody
- Mega-9(10)
nonanoyl(decanoyl)-N-methylglucamide
- Mr
apparent molecular mass
- PMSF
phenylmethyl-sulfonyl fluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
9.
The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.Abbreviations EDTA
ethylenediaminetetraacetic acid
- FMN
flavinmononucleotide
- PMSF
phenylmethylsulfonylfluoride
- Pr(Pfr)
red(far-red)-absorbing form of phytochrome
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
10.
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans. 相似文献
11.
R. C. Gupta E. F. Young D. G. Ferguson E. G. Kranias 《Molecular and cellular biochemistry》1991,102(2):165-172
A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 Mr protein. Maximal phosphorylation of the 55,000 Mr protein occurred in the presence of Mg2+-ATP. Addition of cAMP, cGMP, Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 Mr protein. However, in the presence of Ca2+/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg2+-NTPase activity. These results indicate that a protein with Mr 55,000 may be involved in the regulation the Mg2+-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg
Hemoglobin
- GAR
Goat Anti-Rabbit antibody
- SR
Sarcoplasmic Reticulum
- NTP
Nucleoside Triphosphate
- TCA
Trichloroacetic acid
- PAGE
Polyacrylamide gel electrophoresis 相似文献
12.
Peptide-transport proteins, intrinsic to the epithelial plasmalemmae of the scutella of germinating barley (Hordeum vulgare L.) embryos, have been selectively labelled with p-chloro-[203Hg]mercuribenzenesulphonate using both a substrate-screening technique and a procedure developed to label exclusively vicinal dithiol groups, which were shown previously (Walker-Smith and Payne, 1983, FEBS Lett. 160, 25–30) to be essential components of the peptide-transport system. After radioactive labelling, proteins from the scutellar membranes have been solubilised with lithium diiodosalicylate plus sodium dodecyl sulphate and separated by using polyacrylamide gel electrophoresis. Fluorography and silver staining of these gels has for the first time allowed identification of two presumptive components of the peptide-transport system. These components only become detectable in an extract of the scutellar epithelia after 15 h imbibition, concomitant with a dramatic increase in peptide-transport activity, and they remain present at least 3 d after the onset of germination. [35] Methionine was shown to be incorporated into these proteins between 15–20 h after imbibition, but its incorporation during a similar 5 h period into scutella isolated after 3 d was undetectable, implying a slow turnover of these proteins during the later stages of germination.Abbreviations Ala2, Ala3
dialanine, trialanine
- CHAPS
3-((3-cholamidopropyl) dimethylammonio)-1-propanesulphonate
-
p-CMBS
p-chloromercuribenzenesulphonic acid
- NEM
N-ethylmaleimide
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
13.
Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.Abbreviations DAB
diamino benzidine tetrachloride
- FITC
fluorescein isothiocyanate
- kDa
kilodalton
- PBS
phosphate-buffered saline
- PP
phloem polypeptide(s)
- SDS-PAGE
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
14.
Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent KmS for -ketoglutarate, NADPH and NH
inf4
sup+
are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA
Ethylenediamine Tetraacetic Acid
- Tris
Tris(hydroxymethyl)aminomethane
- Bis-tris
bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane
- TRITON X-100
iso-octylphenoxypoly-ethoxyethanol
-
pHMB
p-Hydroxymercuribenzoic acid 相似文献
15.
High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli 总被引:1,自引:0,他引:1
As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in
converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant
enzyme was purified and biochemically characterized. The apparent K
m values of the enzyme for 1,3-propanediol and NAD+ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C. 相似文献
16.
Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA
enzyme-linked immunsorbent assay
- kDa
kilodalton
- mAb
monoclonal antibody
- Pfr
far-red-absorbing form of phytochrome
- Pr
red-absorbing form of phytochrome
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- (A)
difference in absorbance (A
665
Pr
–A
730
Pr
)-(A
665
Pfr
–A
730
Pfr
)
- Ar/Afr
spectral change ratio (SCR)
- max
mole fraction of Pfr following saturating red light 相似文献
17.
G. Burkhardt G. Wegener 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(4):261-271
Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE
diethylaminoethyl
- EDTA
ethylenediamine tetraacetate
- EGTA
ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid
-
M
r
relative molecular mass
- NMR
nuclear magnetic resonance
- PAGGE
polyacrylamide gradient gel electrophoresis
- Pi
morganic phosphate
- SDS
sodium dodecylsulphate
- TRIS
tris(hydroxymethyl)-aminomethane
-
V
max
maximum activity 相似文献
18.
Arthur M. Edelman Wei-Hsung Lin Donna J. Osterhout Mark K. Bennett Mary B. Kennedy Edwin G. Krebs 《Molecular and cellular biochemistry》1990,97(1):87-98
Brain type II Ca2+/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca 2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2+-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
- EGTA
Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid
- DTT
Dithiothreitol
- LC20
Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain
- LC17
Gizzard Smooth Muscle, 17 kDa Myosin Light Chain
- H Chain
Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain
- TPCK
L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone
- MOPS
3-(N-morpholino) Propanesulfonic Acid 相似文献
19.
Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.Abbreviations DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- FPLC
fast protein liquid chromatography
- kDa
kilodaltons
- PAGE
polyacrylamide gel electrophoresis
- PMSF
phenylmethylsulphonyl fluoride
- RuBPCase
ribulose bisphosphate carboxylase
- SDS
sodium dodecyl sulphate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
20.
Noriko Yokoyama 《Molecular and cellular biochemistry》1995,148(2):123-132
S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn2+. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn2+-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC50 of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn2+.Abbreviations PP1-C
catalytic subunit of type 1 protein phosphatase
- SDS
sodium dodecyl sulfate
- Hepes
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
- PMSF
phenylmethylsulfonyl fluoride
- Mops
4-morpholine propanesulfonic acid
- EDTA
ethylenediaminetetraacetate
- EGTA
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