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1.
1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P1 was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P1 fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg2+, yielded the P1(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P1(S) fraction from E. coli K10 wild type (R+1R+2P+) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P1(S) fractions of two constitutive strains as with the P1(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P1(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn2+ to the incubation mixtures. However, Mn2+ inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [32P]CTP into RNA was overcome by Mn2+. The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P1(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of β-galactosidase by the same P1(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn2+-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.  相似文献   

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3.
The cellular site and characteristics of the phosphorylation of a nucleolus-specific phosphoprotein (molecular weight, 120 000) in mouse ascites tumor cells were studied. The phosphoprotein was strongly labeled with 32P when the isolated nucleoli were incubated with [γ-32P]ATP in vitro. This phosphoprotein, and protein kinase for the protein phosphorylation were both purified from 0.3 M KCl soluble protein fraction of the nucleoli by hydroxylapatite and phosphocellulose column chromatographies. It was found that phosphorylation of the nucleolus-specific phosphoprotein was catalyzed selectively by a guanosine 3:5-monophosphate-dependent protein kinase in the nucleoli and the reaction product was the same phosphoprotein as the substrate used.  相似文献   

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6.
Immature rat uterine nucleoli were isolated and their ability to synthesize RNA in vitro was determined. Estradiol-17β injected intraperitoneally 2 hr prior to killing stimulated rat uterine nucleolar in vitro RNA synthesis both quantitatively and qualitatively. The intraperitoneal administration of cycloheximide as late as 10 min prior to the end of a 2-hr estrogen exposure prevented both the quantitative and qualitative changes stimulated by estrogen. The data suggest that estrogen-stimulated rat uterine nucleolar RNA synthesis requires the continuous synthesis of protein.  相似文献   

7.
《Insect Biochemistry》1987,17(7):939-942
We compared the effects of three treatments (brain extracts, high K+, forskolin) which inhibit JH synthesis by corpora allata of the cockroach Diploptera punctata incubated in vitro. Corpora allata showed a similar developmental sensitivity to all three treatments, with a high level of inhibition in glands of low activity. Inhibition of JH III synthesis by forskolin, high K+ or brain extract was antagonized by mevalonate and farnesoic acid, thus implicating an early biosynthetic step as the target of inhibition. All three treatments were also antagonized by Mn2+, suggesting that the inhibitory mode of action may be dependent upon Ca2+. Inhibition for forskolin and by high K+ was not only reversible, but caused a stimulation of JH synthesis upon removal of the inhibitor, suggesting that more than one regulatory mechanism is affected by these treatments.  相似文献   

8.
A soluble fraction, obtained by extracting E. coli cytoplasmic membrane vesicles with water, transfers radioactivity from [γ-32P]ATP to a protein present in this soluble fraction. The formation of the [32P]phosphoprotein appears to be reversible. Thus the protein can transfer its 32P to ADP to form [32P]ATP, and the phosphate on the protein can exchange with the phosphate of ATP. Preliminary evidence indicates that the phosphate moiety is linked to a histidine residue of the protein. The Mn2+ and ATP dependencies of [32P]phosphoprotein formation are almost identical to the diglyceride kinase reaction previously reported in intact membrane vesicles. Although indirect evidence supports the involvement of the phosphoprotein in the diglyceride kinase reaction, the soluble fraction catalyzes only a slow formation of [32P]phosphatidie acid from [γ-32P]ATP and α,β-diglyceride.  相似文献   

9.
RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg2+ and Mn2+ will support significant rates of activity in vitro against natural RNAs, with Mn2+ being preferred. Both Mg2+ and Mn2+ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni2+ and Zn2+ permitted low levels of activity, while Ca2+, Co3+, Cu2+, and Fe2+ did not. A mutation to one of the residues known to chelate Mg2+, D346C, led to almost complete loss of activity dependent on Mg2+; however, the activity of the mutant enzyme was fully restored by the presence of Mn2+ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO4, implying that RNase E relies on Mg2+ exclusively in vivo.  相似文献   

10.
Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules, called autoinducers. LuxS and Pfs catalyze synthesis of the quorum-sensing signaling molecule autoinducer 2 (AI-2), which has been shown to control a variety of cellular processes. We studied the cloning, expression, and purification of LuxS and Pfs from Streptococcus suis Serotype 2 strain HA9801 (SS2); the two enzymes gave an apparent single protein band, and revealed a molecular mass of 21.74 and 28.44 kDa on an SDS-PAGE, respectively. Expressed and purified LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH). The reaction products were able to induce luminescence of Vibrio harveyi BB170, clearly demonstrating that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. Optimum pH and temperature for biosynthesis AI-2 in vitro were 8.0 and 37 °C, respectively. Biosynthesis AI-2 in vitro was stimulated by Cr3+, Al3+, and Ba2+and was inhibited by Fe2+ and Ni2+, respectively. It was strongly inhibited by Hg2+, Cu2+, and Mn2+, while enzyme activity was not affected by Li+, Mg2+, and Zn2+. In this study, we cloned, expressed, and purified LuxS and Pfs, identified the pathway of AI-2 synthesis in SS2, and analyzed the impact factor of AI-2 synthesis in vitro, which provided a solid basis for future research concerning the role of AI-2 in SS2.  相似文献   

11.
The activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, is differentially distributed in various regions of the rat brain. The cerebellum possessed the highest enzyme activity of the eight regions studied. The cerebral cortex and the midbrain also exhibited high 5-aminolaevulinate synthase activity; the septum, hypothalamus, thalamus, amygdala and the hippocampus possessed much lower enzyme activity. However, the total porphyrin and haem contents of the different brain segments did not vary greatly. Mn2+, when administered subcutaneously to rats, effectively inhibited the activity of 5-aminolaevulinate synthase in the cerebellum, midbrain and cerebral cortex; however, repeated injections of the metal ion neither decreased the haem and porphyrin contents of the brain nor induced haem oxygenase activity. Mn2+ was not an effective inhibitor of 5-aminolaevulinate synthase activity in vitro. On the other hand, studies carried out with the liver in vivo suggested that Mn2+ may alter the turnover rate of cellular haem and haemoproteins. In that event, it is likely that the inhibition of 5-aminolaevulinate synthase by Mn2+ was in part a result of the inhibition of protein synthesis by the metal ion. It is postulated that the haem and porphyrin contents of the brain are maintained at a steady-state level, due in part to the refractoriness to inducers of the regulatory mechanism for haem catabolic enzymes and in part to the ability of the organ to utilize haem precursors derived from extraneuronal sources.  相似文献   

12.
13.
M. Perl 《Phytochemistry》1981,20(8):1791-1793
An enzyme which splits reduced NAD has been partially purified from pea (Pisum sativum, Kelvedon Wonder) seeds. The activity requires orthophosphate and the products are ADP and probably NMN (dihydro NMN?). The enzyme splits the NADH2 at the pyrophosphate bond and incorporates the phosphate into the AMP residue. NAD, NADP or NADPH2 could not replace NADH2. The enzyme is unstable during storage, is activated by Mg2+ and by Mn2+, and inhibited by Ca2+. K+, Li+ and NH4+ have no effect. The possible role of this enzyme in the synthesis of ATP in seeds at the early stage of germination is discussed.  相似文献   

14.
Alkalophilic Bacillus no. C-3 isolated from soil produced 5′-nucleotidase (EC 3.1.3.5) extracellularly when cultured in a medium containing Mn2+. The unique point of enzyme production is that the enzyme was produced well in the medium containing a rather high concentration of Mn2+, in spite of a small difference in growth. The optimum concentration of Mn2+ for the enzyme production was 10 mM and over. Mn2+ could not be replaced by other divalent cations when added singly. In the presence of 10 mM Mn2+, the enzyme production was repressed by the addition of 0.5 mM phosphate to the medium. The course of the enzyme production closely paralleled the increase in growth. The optimum pH for the enzyme activity was 9.2–9.5, and KHCO3-K2CO3 buffer was suitable for the enzyme.  相似文献   

15.
An investigation of phosphoprotein phosphatase activity in rabbit liver membrane using 32P-labeled histone and phosphorylase as substrates has shown that the activity is inhibited by preincubation in a phosphorylating system containing ATP or GTP as well as in the presence of physiological concentrations of inorganic phosphate. Kinetics of inhibition by both ATP and inorganic phosphate are noncompetitive. Phosphatase activity has a broad pH optimum of 7.5–9.0 and is not stimulated by Mg2+, Mn2+, or Zn2+.  相似文献   

16.
Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg2+ and Mn2+, Mn2+ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.  相似文献   

17.
We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn2+, PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co2+ PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn2+ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co2+ was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively.  相似文献   

18.
In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+. The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn2+ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn2+ efflux. We propose that Hip1p is involved in Mn2+ transport, carrying out a function related to Mn2+ export.  相似文献   

19.
Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn2+-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.  相似文献   

20.
The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders.  相似文献   

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