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11.
Horn A Kreusch S Bublitz R Hoppe H Cumme GA Schulze M Moore T Ditze G Rhode H 《Proteomics》2006,6(2):559-570
A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags. 相似文献
12.
Hayden D Jennings A Müller C Pascoe D Bublitz R Webb H Breithaupt T Watkins L Hardege J 《Hormones and behavior》2007,52(2):162-168
Experiments were conducted to investigate the sex-specific differences to feeding responses of the shore crab Carcinus maenas throughout the year. Results demonstrate that female shore crabs exhibit stronger feeding responses than males throughout the year with a significantly reduced feeding response in males during the summer months' reproductive season. We also studied the possible function(s) of the moulting hormone, 20-hydroxyecdysone (Crustecdysone) that has been described as a potential female-produced sex pheromone to initiate male reproductive behaviour in a number of crustaceans. We recently presented evidence that for shore crabs this is not the case and now show that the steroid is instead functioning as a sex-specific feeding deterrent protecting the moulting 'soft' female crabs. Whilst male shore crabs were deterred from prey (Mytilus edulis) and synthetic feeding stimulants glycine and taurine when these feeding stimulants were spiked with crustecdysone, intermoult female crabs were significantly less affected and rarely deterred from feeding. This sex specificity of the moulting hormone, in combination with the female sex pheromone, which has no anti-feeding properties, ensures that male crabs mate with soft-shelled, moulted females rather than engage in cannibalism, such as found frequently in cases when soft-shelled females are exposed to intermoult females. 相似文献
13.
Fletcher Nichola Terschak John A. Bartels-Hardege Helga D. Bublitz Ralf Schirrmacher Paula Hardege Jorg D. 《Aquatic Ecology》2022,56(2):419-427
Aquatic Ecology - The reproduction of many brachyuran crustaceans involves the formation of mating pairs often around the time of the female moult with attraction of a sexual partner and mating... 相似文献
14.
E Hoffmann-Blume M B Garcia Marenco H Ehle R Bublitz M Schulze A Horn 《European journal of biochemistry》1991,199(2):305-312
1. Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20-34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135,000 x g for 60 min. whereas the IAP of rodents is soluble in the range of 60-72% of the total IAP. 2. For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X-114 phase partitioning, phenyl-Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and papain digestion. 3. More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns-anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction. 4. MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure. 5. The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed. 相似文献
15.
Various concentrations of Tergitol NP-10 stimulate mannose-6-phosphatase and glucose dehydrogenase to the same extent in untreated rat liver microsomes. Thus, the latency of glucose dehydrogenase may be used as an alternative to mannose phosphatase as a measure of the integrity of the microsomal membrane. The advantage of using glucose dehydrogenase rather than mannose phosphatase to monitor microsomal integrity is that NADH is more easily measured than Pi. 相似文献
16.
Bublitz R Kreusch S Ditze G Schulze M Cumme GA Fischer C Winter A Hoppe H Rhode H 《Proteomics》2006,6(13):3909-3917
A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples. 相似文献
17.
Mette Laursen Maike Bublitz Karine Moncoq Claus Olesen Jesper Vuust M?ller Howard S. Young Poul Nissen J. Preben Morth 《The Journal of biological chemistry》2009,284(20):13513-13518
We have determined the structure of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) in an E2·Pi-like form
stabilized as a complex with , an
ATP analog, adenosine 5′-(β,γ-methylene)triphosphate
(AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å
resolution leads to a significantly revised model of CPA binding when compared
with earlier reports. It shows that a divalent metal ion is required for CPA
binding through coordination of the tetramic acid moiety at a characteristic
kink of the M1 helix found in all P-type ATPase structures, which is expected
to be part of the cytoplasmic cation access pathway. Our model is consistent
with the biochemical data on CPA function and provides new measures in
structure-based drug design targeting Ca2+-ATPases, e.g.
from pathogens. We also present an extended structural basis of ATP modulation
pinpointing key residues at or near the ATP binding site. A structural
comparison to the Na+,K+-ATPase reveals that the
Phe93 side chain occupies the equivalent binding pocket of the CPA
site in SERCA, suggesting an important role of this residue in stabilization
of the potassium-occluded E2 state of Na+,K+-ATPase.The Ca2+-ATPase from sarco(endo)plasmic reticulum of rabbit
skeletal muscle
(SERCA,5 isoform 1a)
is a thoroughly studied member of the P-type ATPase family
(1). SERCA possesses 10
transmembrane helices (M1 through M10) with both the N terminus and the C
terminus facing the cytoplasmic side and three cytoplasmic domains, inserted
in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain)
(2). The enzyme mediates the
uptake of Ca2+ ions into the lumen of the sarcoplasmic reticulum
(SR) after their release into the cytoplasm through calcium release channels
during muscle contraction (3).
SERCA, plasma membrane Ca2+-ATPase, and a third, Golgi-located
secretory pathway Ca2+-ATPase are important factors in calcium and
manganese homeostasis, transport, signaling, and regulation
(4,
5).Crystal structures of all major states in the reaction cycle of SERCA have
been determined. These include the Ca2E1·ATP
state (6,
7) with high affinity
Ca2+ binding sites accessible from the cytoplasmic side of the SR
membrane, the calcium-occluded
transition state (6), the open
E2P state with luminal facing ion binding sites that have low affinity for
Ca2+ and high affinity for protons
(8) and the proton-occluded
H2–3E2[ATP] state with a bound modulatory ATP
(9). This considerable amount
of structural information has turned the Ca2+-ATPase into a
valuable model system for studies on structural rearrangements that take place
during the catalytic cycle of P-type ATPases. SERCA is considered a promising
drug target in medical research, with a particular focus on prostate cancer
and infectious diseases. Several compounds have already been shown to bind and
inhibit SERCA by stabilizing the enzyme in a particular conformational state.
Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)
hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri
(methylisothiouronium)benzene stabilizes an E1-P-like conformation
(10–13).
CPA is a toxic indole tetramic acid first isolated from Penicillium
cyclopium (14) and later
found to be produced by Aspergillus versicolor and Aspergillus
flavus. Like TG, CPA specifically binds to and inhibits SERCA with
nanomolar affinity (15).
Indeed, CPA is widely used in biochemical and physiological studies on
Ca2+ signaling and muscle function, where it causes Ca2+
store depletion due to specific inhibition of Ca2+ reuptake by
SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA
(16), but recent crystal
structures have shown a distinct site of interaction
(17,
18). Despite these structural
insights, a previously demonstrated magnesium dependence of CPA binding
(19) remained unexplained, and
opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural
derivatives have been isolated from bacterial and fungal species (reviewed in
Ref. 20). Tetramic acids
possessing a 3-acyl group have the ability to chelate divalent metal ions. For
instance, tenuazonic acid from the fungus Phoma sorghina has been
shown to form complexes with Ca2+ and Mg2+
(21), as well as heavier
metals such as Cu(II), Ni(II), and Fe(III)
(22).Previously published crystallographic structures of the SERCA·CPA
complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed
calcium access channel of SERCA. However, the structures did not reveal a role
for magnesium, and the orientation of CPA within this binding site differed in
the two studies (17,
18). To address these
ambiguities, we have determined the crystal structure of SERCA in complex with
, AMPPCP (an ATP analog), and
Mn2+·CPA. The structure reveals novel insight into CPA
binding, which we find to be mediated by a divalent cation, as demonstrated by
means of the anomalous scattering properties of Mn2+. Further and
improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in
light of our new findings, also revealed a strong plausibility for a magnesium
ion bound at this site. Furthermore, we find a new configuration of the bound
AMPPCP nucleotide, addressing the modulatory role of ATP binding to the
E2·Pi occluded conformation of SERCA. 相似文献
18.
The levels of nicotinamide nucleotides in liver microsomes and their possible significance to the function of hexose phosphate dehydrogenase. 总被引:1,自引:0,他引:1 下载免费PDF全文
The concentrations of NAD and NADP have been determined in detergent extracts of washed rat liver microsomes. Precautions were taken during the preparation of the microsomes to remove nicotinamide nucleotides from their external surface both by hydrolysis by nucleotide pyrophosphatase (EC 3.6.1.9) and by washing them three times in 0.15 M-Tris/HCl, pH 8.0, to remove soluble proteins which bind these nucleotides. The mannose phosphatase was essentially completely latent, indicating that the microsomes were intact. Assuming these nucleotides are in the cisternae of the microsomes, the concentrations in the cisternae are 240 +/- 25 microM-NAD and 55 +/- 12 microM-NADP. These levels of nucleotides are compatible with both the glucose:NAD+ and the glucose 6-phosphate:NADP+ oxidoreductase activities of hexose phosphate dehydrogenase (EC 1.1.1.47). Since the organ and subcellular distributions of this dehydrogenase and glucose-6-phosphatase are similar, and Pi stimulates the glucose:NAD+ oxidoreductase activity, it is proposed that the combined action of these two enzymes leads to the reduction of both coenzymes in the lumen of the endoplasmic reticulum. A modification of the colorimetric method of Nisselbaum & Green [(1969) Anal. Biochem. 27, 212-217] for the determination of NADP+ is described. Colour formation is linear with the concentration of NADP+ and is sensitive to less than 0.3 nmol of NADP+. 相似文献
19.
J. Miersch G. -J. Krauss K. Grancharov F. Bublitz N. Spassovska E. Golovinsky 《Biologia Plantarum》1986,28(3):174-179
The pyruvic acid thiosemicarbazone was found to inhibitin vitro the soluble ornithine carbamoyl transferase as well as the enzyme in a mitochondria-rich cell fraction. For proving the inhibition
of ornithine carbamoyl transferase in mitochondria-rich fraction a new method for DEAE-thin-layer chromatography of radiocarbon
labelled citrulline and ornithine was used. The mitochondria prepared by discontinuous sucrose density gradient centrifugation
are physiologically active and oxidized succinate under state 3 conditions. The respiratory control ratio was determined to
3.3. The results obtained show that this analogue of pyruvic acid is an inhibitor of ornithine carbamoyl transferase.
In memoriam of Prof. Dr. H. Reinbothe 相似文献
20.
Clark Bublitz 《Biochemical and biophysical research communications》1981,98(3):588-594
Washed rat liver microsomes contain a 6-phosphogluconate dehydrogenase (E.C.1.1.1.44) which is partially extracted by a nonionic detergent. This enzyme can be distinguished from the 6-phosphogluconate dehydrogenase in the microsomal supernatant fraction by separations in polyacrylamide gels by electrophoresis or isoelectric focusing. The possible significance of this enzyme in microsomes is discussed. 相似文献