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1.
A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an
absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as
determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two
subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)6 subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony
double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined
by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins. 相似文献
2.
S.I. Ortiz-Miranda J.A. Lasalde P.A. Pappone M.G. McNamee 《The Journal of membrane biology》1997,158(1):17-30
We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys
residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold,
respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open
time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold.
No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested.
However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional
conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the
α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate
and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the
α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating,
and that mutations in multiple subunits act additively to increase channel open time.
Received: 27 September 1996/Revised: 28 January 1997 相似文献
3.
Hagner-Holler S Kusche K Hembach A Burmester T 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2005,175(6):445-452
Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea,
hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been
known from the ’lower’ Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative
partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa.
Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the α-type
of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to α and γ-subunits most likely
at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further
suggest that the divergence of β-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA)
in the malacostracan stemline, while α- and γ-type subunits separated 258 MYA, and pseudohemocyanins and γ-subunits 210 million
years ago. 相似文献
4.
In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of α′ and β subunits of β-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the α′ subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of α′ subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the α and β subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD. 相似文献
5.
Kubiński K Domańska K Sajnaga E Mazur E Zieliński R Szyszka R 《Molecular and cellular biochemistry》2007,295(1-2):229-236
Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears
to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic
(α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four
active forms of CK2, composed of αα′ββ′, α2ββ′, α′2ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic
unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing
single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation
experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits
in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast
protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity. 相似文献
6.
7.
Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors 总被引:3,自引:0,他引:3
A. Terakita H. Takahama T. Hariyama T. Suzuki Y. Tsukahara 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1998,183(4):411-417
In crayfish photoreceptor cells, Gq-type G-protein plays a central role in the phototransduction pathway, and the translocation
of Gqα has been proposed as one of the molecular mechanisms to control photoreceptor sensitivity. We here investigated β subunit
of Gq and its localization profiles under various light conditions in the crayfish photoreceptor cells to understand the functional
characteristic of visual Gq in the phototransduction pathway. An immunoprecipitation experiment was performed using an anti-Gqα
antibody and a thiol-cleavable crosslinker. A 39 kDa protein was co-immunoprecipitated with Gqα, but not by irradiation, in
the presence of GTPγS. The partial amino acid sequence of the 39 kDa protein was similar to Gβe in Drosophila photoreceptors, indicating that the crayfish Gβ which combines with Gqα is a Gβe homologue. Immunohistochemical and immunoblot
analyses revealed that the amount of the Gβ decreased in the rhabdomeric membranes and increased in the cytoplasm in the light,
compared with that in the dark. The profile of the translocation was similar to that reported for Gqα. Since both α and βγ
subunits are necessary for G-proteins to be activated by rhodopsin in the rhabdom, the light-modulated translocation of a
Gβe homologue possibly controls the amount of Gq which can be activated by light-stimulated rhodopsin.
Accepted: 27 June 1998 相似文献
8.
The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their
domain B, a distinct domain that protrudes from the regular catalytic (β/α)8-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed
by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional
structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different
forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in
the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase,
cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B
of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable
similarity with (β/α)8-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in
turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only
parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family.
Received: 4 December 1996 / Accepted: 13 March 1997 相似文献
9.
The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the
evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary
distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58
residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)8-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one
based on the alignment of the substantial continuous part of the (α/β)8-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared
brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes,
which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases.
This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases,
especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya,
Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence
regions may indeed constitute the ``sequence fingerprints' of a given α-amylase.
Received: 3 June 1998 / Accepted: 20 August 1998 相似文献
10.
Masłyk M Kochanowicz E Zieliński R Kubiński K Hellman U Szyszka R 《Molecular and cellular biochemistry》2008,312(1-2):61-69
Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid
sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification
and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant
CK2α (K
m 0.35 μM) and CK2α′ (K
m 0.18 μM) as well as CK2 holoenzyme (K
m 1.1 μM). Different K
m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor
Svf1. Reconstitution of α′2ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit.
This effect was not observed in the case of α2ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments).
Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and
TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation
of cell survival pathways. 相似文献
11.
Jones AK Marshall J Blake AD Buckingham SD Darlison MG Sattelle DB 《Invertebrate neuroscience : IN》2005,5(3-4):147-155
The cloning, sequencing and functional expression of Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dβ1 and 92% identity with the Locusta migratoria β1, non-α subunits but only 38% identity to Sgα1 (also referred to as αL1), a previously cloned S. gregaria nAChR α-subunit. When expressed in Xenopus laevis oocytes, Sgβ1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgα1 and Sgβ1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgα1 alone. We conclude that either Sgβ1 does not co-assemble with Sgα1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system. 相似文献
12.
Kolaiti RM Baier A Szyszka R Kouyanou-Koutsoukou S 《Marine biotechnology (New York, N.Y.)》2011,13(3):505-516
Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly
serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme
is generally composed of two catalytic (α and/or α′) and two regulatory (β) subunits, forming a differently assembled tetramer.
The free and catalytically active α/α′ subunits can be present in cells under some circumstances. We present here the isolation
of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and
the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme
presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz),
and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs. 相似文献
13.
Yanan Cao Yaru Wang Kun Meng Yingguo Bai Pengjun Shi Huiying Luo Peilong Yang Zhigang Zhou Zhifang Zhang Bin Yao 《Applied microbiology and biotechnology》2009,83(5):875-884
A novel α-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass
of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino
acid sequence showed highest identity (69%) with an α-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60°C and pH 4.0 and
was stable at pH 3.0–12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline
proteases. The enzyme K
m values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared
with the commercial α-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant α-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold
more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity
of Aga-F75 make it potentially useful in the food and feed industries. 相似文献
14.
Kamila Rzeznicka Sebastian Schätzle Dominique Böttcher Joachim Klein Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2010,85(5):1417-1425
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly
in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator
gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration
and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic
ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity
was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest
stability at 4°C in thermal inactivation studies between 4°C and 50°C. 相似文献
15.
Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin
Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture.
The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of
(αβ)6 hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and
the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm)
and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit
is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps
contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption
maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the
fluorescence emission maximum at 575 nm. 相似文献
16.
Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr
43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem
Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices,
composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are
connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural
arrangements of this subunit inside the V1- and V1VO ATPase. 相似文献
17.
Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels.
To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal
calcium channels (α1A, α1B, α1C, and α1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift
in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the
activation-curve. Block followed 1:1 kinetics and was most pronounced for α1C, followed by α1E > α1A > α1B channels. In contrast, the change in activation-gating was most dramatic with α1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple
model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking
site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium
with calcium reduced both the degree of nickel block and the maximal effect on gating for α1A channels, but increased the nickel blocking affinity for α1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α1A, as coexpression with β2a or with β4 resulted in larger current-voltage shifts than those observed in the presence of β1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the
three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest
that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively
and quantitatively among individual subtypes and subunit combinations.
Received: 12 October 1995/Revised: 17 January 1996 相似文献
18.
Cho HY Seo MJ Park JU Kang BW Kim GY Joo WH Lee YC Cho YS Jeong YK 《Journal of microbiology (Seoul, Korea)》2011,49(6):1018-1021
A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific
for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, β, and γ chains in that order.
The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic
enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results
provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus. 相似文献
19.
20.
Parrinello N Vizzini A Arizza V Salerno G Parrinello D Cammarata M Giaramita FT Vazzana M 《Cell and tissue research》2008,334(2):305-317
A tumor necrosis factor-alpha (TNFα)-like gene from Ciona intestinalis (CiTNFα-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation.
An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from
positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a
predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein
and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFα-like cytokine
was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels
were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive
hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human
TNFα epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate
supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNFα-like protein therefore appeared
to be modulated by the LPS challenge.
This work was supported by a research grant from the Italian Ministry of University and Scientific Research (PRIN 2006 to
N. Parrinello), co-funded by the University of Palermo. 相似文献