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61.
Transport of amino acids into the oestrogen-primed uterus. Enhancement of the uptake by a preliminary incubation 总被引:1,自引:0,他引:1
1. Preincubation of the immature rat uterus under physiological conditions was found to increase its subsequent ability to transport alpha-aminoisobutyric acid, l-proline, l-alanine and 1-aminocyclopentanecarboxylic acid. Uptakes of l-valine, l-phenylalanine and l-leucine were not affected. With alpha-aminoisobutyric acid, a doubling of the uptake could be obtained after a 3-5h preincubation period. Uteri from oestradiol-primed rats gave increases similar to those found in tissues from untreated animals. In both cases the preincubation increased the V(max.) of alpha-aminoisobutyric acid uptake but did not affect the K(m). 2. The conditions during the preincubation period determined the increase in subsequent uptake of alpha-aminoisobutyric acid. No increase in uptake was found if the preincubation was carried out at 1 degrees C, in the presence of cyanide or dinitrophenol, under anaerobiosis or with a concentration of puromycin that depressed incorporation of l-leucine into protein by 95%. The puromycin was also shown to prevent the increase in V(max.) normally found after the preincubation period. In addition, no increase was found if Na(+) was omitted from the preincubation medium. Other inorganic ions had smaller effects. 3. The uptake of alpha-aminoisobutyric acid by uteri before and after a preincubation period showed the same general patterns of sensitivity to competitive inhibitors, K(+), pH, temperature and 2,4-dinitrophenol. 4. The results suggest that the preincubation leads to an increase in a protein component of the ;A system' for amino acid transport in the uterus, and that metabolic energy is required for the reactions involved. 相似文献
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63.
Evolutionary conservation of the substrate-binding cleft of phosphoglycerate kinases 总被引:6,自引:0,他引:6
The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve beta-sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3-phosphoglycerate-binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner-loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner-loop region. Trypanosome PGK contains a polypeptide segment in its N-terminal domain that is transposed in comparison with the other species. 相似文献
64.
In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive. 相似文献
65.
Austen F. Riggs Claire K. Riggs 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(6):1071-1075
Background: The crystallographic structure of the gigantic hemoglobin (erythrocruorin) of the annelid worm, Lumbricus terrestris, provides a molar mass of 3.6 MDa for the hexagonal bilayer structure. Prior to this determination, some light-scattering and ultracentrifugal measurements indicated higher masses: 4.1–4.4 MDa. Values of 3.6 MDa were attributed to dissociation or subunit loss. However, early electron microscopy of the giant hemoglobin from a related annelid, Eumenia crassa by Öster Levin, showed that the hexagonal bilayer molecules were present mostly as oligomers; few were monomeric. Methods: Measurements by light-scattering of solutions of Lumbricus hemoglobin resolved by size-exclusion chromatography have been used to determine the weight-average molar mass of self-associating proteins. The X-ray structure has been re-examined. Results: Our measurements show that both 3.6 MDa monomers and self-association products are present as a mixture. Analysis of the X-ray structure indicates several different kinds of monomer–monomer interactions. Conclusions: We propose that the measured masses of Lumbricus hemoglobin as high as 4.4 MDa, result from oligomerization. These masses would result from the presence of an array of oligomers of various sizes together with monomers of 3.6 MDa. Furthermore, several different kinds of monomer–monomer interactions are clearly evident in the X-ray structure as well as in solution. General significance: The results demonstrate that self-association of monomers of the hemoglobin of Lumbricus terrestris explains the high molar masses of 4.1–4.4 MDa previously observed. 相似文献
66.
Guidenn Sulbarán Antonio Biasutto Lorenzo Alamo Claire Riggs Antonio Pinto Franklin Méndez Roger Craig Raúl Padrón 《Biophysical journal》2013,105(9):2114-2122
Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism. 相似文献
67.
Farnsworth ML Miller RS Pedersen K Lutman MW Swafford SR Riggs PD Webb CT 《PloS one》2012,7(3):e32729
Outbreaks of avian influenza in North American poultry have been linked to wild waterfowl. A first step towards understanding where and when avian influenza viruses might emerge from North American waterfowl is to identify environmental and demographic determinants of infection in their populations. Laboratory studies indicate water temperature as one determinant of environmental viral persistence and we explored this hypothesis at the landscape scale. We also hypothesized that the interval apparent prevalence in ducks within a local watershed during the overwintering season would influence infection probabilities during the following breeding season within the same local watershed. Using avian influenza virus surveillance data collected from 19,965 wild waterfowl across the contiguous United States between October 2006 and September 2009 We fit Logistic regression models relating the infection status of individual birds sampled on their breeding grounds to demographic characteristics, temperature, and interval apparent prevalence during the preceding overwintering season at the local watershed scale. We found strong support for sex, age, and species differences in the probability an individual duck tested positive for avian influenza virus. In addition, we found that for every seven days the local minimum temperature fell below zero, the chance an individual would test positive for avian influenza virus increased by 5.9 percent. We also found a twelve percent increase in the chance an individual would test positive during the breeding season for every ten percent increase in the interval apparent prevalence during the prior overwintering season. These results suggest that viral deposition in water and sub-freezing temperatures during the overwintering season may act as determinants of individual level infection risk during the subsequent breeding season. Our findings have implications for future surveillance activities in waterfowl and domestic poultry populations. Further study is needed to identify how these drivers might interact with other host-specific infection determinants, such as species phylogeny, immunological status, and behavioral characteristics. 相似文献
68.
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70.
Silvia A. Arredondo Tiffany F. Chen Austen F. Riggs Hiram F. Gilbert George Georgiou 《The Journal of biological chemistry》2009,284(36):23972-23979
The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly3-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.Disulfide bonds are critical for the proper folding and structural stability of many exocytoplasmic proteins. The Dsb family of thiol:disulfide oxidoreductase enzymes catalyzes oxidative protein folding in the periplasm of Escherichia coli by means of two independent pathways (1–3). In the DsbA-DsbB oxidation pathway, DsbA, a very strong oxidant, catalyzes the formation of disulfide bonds on newly translocated proteins (4). The DsbA disulfide is rapidly recycled by DsbB, a membrane protein that transfers electrons from DsbA onto quinones (5–7). In the DsbC-DsbD isomerization pathway, non-native disulfides are reduced or rearranged by DsbC. DsbC is maintained in a reduced, catalytically active state via the transfer of electrons from the inner membrane protein DsbD that in turn accepts electrons from thioredoxin 1 and ultimately from NADPH (via thioredoxin reductase) within the cytoplasm (8, 9). Large kinetic barriers keep the oxidation and isomerization pathways isolated, preventing the establishment of a futile cycle of electron transfer. Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 103–107-fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11, 12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an α-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein. The inner surface of the resulting cleft is patched with uncharged and hydrophobic residues suggesting an important role in the binding of substrate proteins. The active sites comprising the sequence Cys98-Gly99-Tyr100-Cys101 in each of the monomeric subunits are located in the arms of the “V” facing each other (13). Isomerization involves an attack onto a substrate disulfide by Cys98 resulting in the formation of a mixed disulfide, which then is resolved by either another cysteine from the substrate or by Cys101 from DsbC (14, 15). Besides its isomerase activity, DsbC is also known to display chaperone activity preventing protein aggregation during refolding (16). In E. coli, disulfide bond isomerization is the limiting step in the oxidative folding of many heterologous proteins that contain multiple cysteines. Overexpression of DsbC has been shown to enhance the yield of proteins such as human nerve growth factor, human tissue plasminogen activator (tPA)2 and immunoglobulins (17–19).DsbC is topologically analogous to the eukaryotic protein-disulfide isomerase (PDI). The structural similarities between the two enzymes may have resulted from convergent evolution by thioredoxin-like domain replication in the case of PDI and domain recruitment in DsbC (20, 21). PDI comprises two thioredoxin-like catalytic domains (a and a′) separated by two non-catalytic domains (b and b′), in addition to a c domain (22). In PDI, the catalytic domains are different and functionally nonequivalent (23). Substrate binding is mediated primarily by the b′ domain; the two catalytic domains, a and a′, can catalyze oxidation of small model peptides indicating that they must also have low substrate binding affinity (24).The DsbC monomer is essentially devoid of RNase A isomerase activity (25). Sun and Wang (44) reported that DsbC with one catalytic site impaired by carboxymethylation is also essentially inactive but, in separate studies, Zapun et al. (26) did not detect cooperativity between the two catalytic sites indicating that they function independently of each other (26). Moreover, unlike PDI, the significance of the putative peptide binding cleft of DsbC on disulfide isomerization has not been ascertained. However, while DsbA or TrxA with a PDI active site dipeptide (CGHC) display very little isomerase activity in vitro and in vivo (27–29), we recently showed that upon fusion to a dimerization region that provides a putative substrate binding surface (the E. coli peptidyl proline isomerase FkpA) they acquire the ability to assist the folding of periplasmically expressed multidisulfide heterologous proteins (30).In the present work, we engineered heterodimer-like covalently linked DsbC derivatives in which one of the catalytic sites has been inactivated (Fig. 1A) or one of the catalytic domains has been entirely removed while maintaining the intact peptide binding cleft (which is normally formed by association of the N-terminal domains of the two monomers) (Fig. 3A). We show that DsbC forced monomers with one functional active site, or with one thioredoxin domain only, display significant isomerization activity. Interestingly, the latter variant is partially reduced in vivo indicating that the presence of both thioredoxin domains is important for the avoidance of protein oxidation by DsbB.Open in a separate windowFIGURE 1.A, protein structure of DsbC, and molecular models of mDsbC-mDsbC and the single active site covalently linked mutants. Dimerization domains are shown in gray, thioredoxin domains in black, and the active sites in white. B, gel filtration FPLC of DsbC and linked variants. Purified proteins were run on a SuperdexTM 200 column in PBS, 10% glycerol buffer.Open in a separate windowFIGURE 3.A, molecular model of mDsbC-dim. Dimerization domains are shown in gray, thioredoxin domain in black, and catalytic site in white. B, gel filtration FPLC of mDsbC-dim as compared with DsbC. Purified proteins were run on a SuperdexTM 200 column in PBS, 10% glycerol buffer. C, MALS measurement of the molar masses of the components of mDsbC-dim together with their hydrodynamic radii. The data show monomeric, dimeric, and tetrameric states. The relative concentrations were determined by the refractive index differences. 相似文献