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71.
Joseph G. Major Jr. Melinda E. Wales John E. Houghton Julie A. Maley Jeffrey N. Davidson James R. Wild 《Journal of molecular evolution》1989,28(5):442-450
Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and
is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated
CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase).
The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported
here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure,
and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the
known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion
protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived
from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB
− cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant
and structurally divergent ATCases. 相似文献
72.
B. E. Slack M. Liscovitch J. K. Blusztajn R. J. Wurtman 《Journal of neurochemistry》1989,53(2):472-481
The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C. 相似文献
73.
Mikio Yoshidomi Takashi Hayashi Koji Abe Kyuya Kogure 《Journal of neurochemistry》1989,53(5):1589-1594
The effects of a new calcium channel blocker, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (KB-2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1, 2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB-2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recirculation. KB-2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB-2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia. 相似文献
74.
S. Romagnoli M. Maddaloni C. Livini M. Motto 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(6):769-775
Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F1 primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F1 hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines. 相似文献
75.
Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase. 相似文献
76.
Techel D. Gebauer G. Kohler W. Braumann T. Jastorff B. Rensing L. 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,159(6):695-706
Summary Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism. 相似文献
77.
Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K m "soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP. 相似文献
78.
Time-Dependent Increase in Protein Phosphorylation Following One-Trial Enhancement in Hermissenda 总被引:1,自引:1,他引:0
Abstract: One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32 PO4 into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning. 相似文献
79.
Takashi Kamaishi Tetsuo Hashimoto Yoshihiro Nakamura Fuminori Nakamura Shigenori Murata Norihiro Okada Ken-ichi Okamoto Makoto Shimizu Masami Hasegawa 《Journal of molecular evolution》1996,42(2):257-263
Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis.
Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139 相似文献
80.
Patricia A. McLenachan Peter J. Lockhart H. Rick Faber Brian C. Mansfield 《Journal of molecular evolution》1996,42(2):273-280
The pregnancy-specific 1-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein. 相似文献