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1.
The nuclear protein kinase NI (NI kinase) was purified from NB-15 mouse neuroblastoma cells by phosphocellulose column and casein affinity column chromatography. The purified NI kinase exhibited (i) an apparent subunit molecular weight of about 37,000, (ii) autophosphorylation, and (iii) insensitivity to inhibition by heparin. When NI kinase was added to heat-treated neuroblastoma nuclei in the presence of [gamma-32P] ATP, two proteins with apparent subunit molecular weights of 11,000 and 10,000 were prominently phosphorylated. Other protein kinases tested including the nuclear protein kinase NII, Type I cAMP-dependent protein kinase, and protein kinase C did not catalyze the phosphorylation of these two proteins. The NI kinase-catalyzed phosphorylation of these two proteins was completely inhibited by 1 mM spermine. In contrast, 10 mM putrescine, 2 mM spermidine, 5 mM arginine, and 10 mM NH4Cl, had no inhibitory effect on this phosphorylation reaction. Our study also indicated that the phosphorylation of the 11,000- and 10,000-dalton proteins occurred in the nuclear matrix fraction but not in heterogeneous nuclear ribonucleoproteins, high mobility group proteins, or histone fractions. We have previously reported that spermine specifically inhibits the endogenous phosphorylation of an 11,000-dalton nuclear protein in various mammalian cell lines (Chen, K. Y., and Verma, R. (1984) Biochem. Biophys. Res. Commun. 118, 710-716). The present study suggests that the 11,000- and 10,000-dalton nuclear proteins may be native substrates of nuclear protein kinase NI and that their phosphorylation can be affected by physiological concentrations of spermine. 相似文献
2.
Toshiro Hara Keikichi Takahashi Mikio Yamamoto Hiroshi Kisaki Hideya Endo 《Biochemical and biophysical research communications》1982,106(1):131-138
The effect of polyamines on the chromatin phosphorylation by endogenous protein kinases was investigated. Polyamines not only selectively stimulated the phosphorylation of chromatin proteins but also concurrently inhibited the phosphorylation of a number of polypeptides. In particular, a 11,000-dalton polypeptide with pI 4.5–5.0 was highly phosphorylated in the absence of polyamines, despite being a minor component whereas the phosphorylation was strongly inhibited in the presence of polyamines. 相似文献
3.
S Levasseur T Poleck G Burke 《Biochemical and biophysical research communications》1985,133(1):354-360
The polyamine, spermine (1-5 mM), when added to rat thyroid cytosol, increases the phosphorylation of a 107 kDa protein 4-fold as analyzed by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE) and autoradiography; spermidine was less effective and putrescine was without effect. Sodium chloride, when tested at equivalent ionic strengths (4-40 mM), did not reproduce the effects of spermine. In addition to stimulating the phosphorylation of a 107 kDa protein, spermine had an apparent biphasic effect on the phosphorylation of 88 and 65 kDa proteins; maximum stimulation of approximately 60-70% was observed at 0.5-2 mM. Both basal and spermine-stimulated protein phosphorylation patterns were identical whether [gamma-32P] ATP or [gamma-32P] GTP was used as phosphate donors. Heparin (1 microgram/ml) reduced spermine-stimulated phosphorylation of the 107 kDa protein by 64%. Phosphorylation of a 107 kDa protein was not restricted to rat thyroid as spermine was found to augment the phosphorylation of 107 kDa protein(s) in mouse and beef thyroid cytosol preparations. 相似文献
4.
Polyamine-stimulated phosphorylation of proteins from corn (Zea mays L.) coleoptiles 总被引:2,自引:0,他引:2
K Veluthambi B W Poovaiah 《Biochemical and biophysical research communications》1984,122(3):1374-1380
The effect of polyamines (spermine, spermidine and putrescine) on in vitro phosphorylation of proteins from corn coleoptiles was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine promoted the phosphorylation of several membrane and soluble proteins and most of the proteins phosphorylated were different from those phosphorylated in the presence of calcium. Spermidine promoted the phosphorylation to a lesser extent and putrescine had very little stimulatory effect. Spermine-promoted phosphorylation of soluble proteins was dependent upon the presence of Mg2+ and was discernible at 100 microM spermine concentration. 相似文献
5.
The phosphorylation of several proteins in isolated nuclei from Pisum sativum L. was stimulated by spermine. Although spermine increased the general protein phosphorylation by 10 to 20%, it increased the phosphorylation of a 47 kilodalton polypeptide by 150%. By comparison other polyamines, spermidine, putrescine, and cadavarine had far less effect on the phosphorylation of the 47 kilodalton or any other polypeptide. Sodium fluoride was able to inhibit the phosphorylation of the 47 kilodalton polypeptide in the control, implying the participation of protein phosphatase(s) in the phosphorylation of nuclear proteins. Spermine stimulated the phosphorylation of the 47 kilodalton polypeptide over the controls, even in the presence of NaF. This result indicates that spermine probably activates a nuclear kinase, a conclusion supported also by thiophosphorylation data. The inability of ethyleneglycol-bis (β-amino-ethyl ether)-N, N′-tetraacetic acid and Compound 48/80, a calmodulin antagonist, to inhibit this spermine stimulated phosphorylation renders improbable any role of calcium and calmodulin in mediating this response. 相似文献
6.
Head Proteins from T-Even Bacteriophages II. Physical and Chemical Characterization 总被引:3,自引:2,他引:1
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Three classes of structural head proteins, having molecular weights of 43,000, 18,000, and 11,000 daltons, were isolated from the T-even bacteriophages and were characterized. Based on electrophoretic studies, the 43,000-dalton class contained one major protein and one (or two) minor components, the 18,000-dalton class contained two protein components, and the 11,000-dalton class contained one major component. The N-terminal residues for the 43,000- and the 11,000-dalton classes were alanine, and the N-terminal residues for the 18,000-dalton class were methionine and alanine. Of the three classes of proteins, the 18,000-dalton proteins were the most acidic, whereas the 11,000-dalton proteins were the most basic. The amino acid composition of the 11,000-dalton class revealed that methionine and cysteine were absent and lysine, histidine, and tryptophan content was higher in the 11,000-dalton class than in the other two classes of proteins. Estimates of the relative number of the three classes of structural proteins were made and indicated that there were between 1,600 and 2,000 subunits of the 43,000-dalton proteins, 100 to 200 of the 18,000-dalton proteins, and 1,000 to 1,500 of the 11,000-dalton proteins. Evidence was presented that the 43,000-dalton proteins and the 11,000-dalton proteins readily formed aggregates with themselves but not with each other. The significance of these interactions to the structure of the T-even phage head was discussed. 相似文献
7.
Early events in the response of fast skeletal muscle to chronic low-frequency stimulation. Polyamine biosynthesis and protein phosphorylation. 总被引:1,自引:1,他引:0
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The concentrations of putrescine, spermidine and spermine and the activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-D) were investigated in fast muscle subjected to chronic low-frequency electrical stimulation. Both ODC and SAM-D activities increased markedly between 18 and 48 h of stimulation. Changes in enzyme activities were followed by phasic elevations in the concentrations of putrescine, spermidine and spermine. Peak levels were reached first by putrescine at 3-4 days, followed by spermidine at about 9 days and then by spermine at about 11 days. A possible relationship was sought between these events and changes produced in vitro in the phosphorylation pattern of cytoplasmic proteins and the total activity of cyclic AMP-dependent protein kinase. However, during the early stages of stimulation, no prominent changes were seen either in the phosphorylation pattern or in the activity of cyclic AMP-dependent protein kinase. These characteristics changed significantly at a later stage (by 12 days of stimulation) and became indistinguishable from those of slow muscle by 3 to 4 weeks of stimulation. 相似文献
8.
Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases. 总被引:1,自引:1,他引:0
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Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases. 相似文献
9.
Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth. 相似文献
10.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1988,972(3):239-248
Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth. 相似文献
11.
Polyamine Regulation of Protein Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta 总被引:1,自引:1,他引:0
Mark J. Birnbaum Wendell L. Combest Timothy J. Bloom Lawrence I. Gilbert 《Journal of neurochemistry》1987,48(3):935-942
An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose-dependent manner (maximum three- to fourfold at 2.0 mM), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE-cellulose at 0.2-0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC50 approximately equal to 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 A, consistent with a Mr of 130,000. Both GTP (Km, 55 microM) and ATP (Km, 34 microM) were utilized as phosphoryl donors (Vmax for ATP being four-fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 microM) strongly enhanced the phosphorylation of three high-molecular-weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system. 相似文献
12.
J J Moore R C Cardaman D W Lundgren 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1984,176(3):313-321
Polyamines are known to have a role in cell proliferation, differentiation, and protein synthesis. During pregnancy, major changes in polyamine levels occur in maternal serum, amniotic fluid, and placental tissue. Polyamine-activated phosphorylation has recently been proposed as a mechanism by which polyamines may regulate metabolic processes in target tissues. Polyamine-activated protein phosphorylation has not been studied in placenta. Homogenate membrane and cytosol fractions from human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of the polyamines, spermine and spermidine, and the diamine, putrescine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, spermine (10(-3) M) significantly (P less than 0.001) stimulated 32P incorporation into phosphoproteins having molecular weights of 55,000 and 105,000. At this concentration spermidine and putrescine failed to stimulate phosphorylation. Half-maximal 32P incorporation was observed with 3.7 +/- 1.25 X 10(-4) M spermine. Polylysine enhanced the phosphorylation of phosphoproteins of the same molecular weight as those enhanced by spermine. Heparin and high Mg2+ inhibited spermine-induced phosphorylation. cAMP and Ca2+ did not stimulate phosphorylation of the spermine-dependent phosphoproteins. Spermine, however, acted as an antagonist for cAMP-dependent phosphorylation of a Mr 45,000 phosphoprotein. 相似文献
13.
A group of chromosomal proteins is specifically released by spermine and loses DNA-binding activity upon phosphorylation. 总被引:1,自引:0,他引:1
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Biologically relevant concentrations as low as 500 microM spermine led to the specific release of chromatin-associated proteins from nuclei of rice (Oryza sativa) seedlings. Using a southwestern technique, it was shown that several of these proteins bind DNA. This affinity was lost upon in organello phosphorylation by an endogenous kinase. The effect of spermine was very specific. Spermidine was far less effective and putrescine was essentially ineffective in releasing these proteins. The most abundant spermine-released protein was shown to be homologous to the maize HMG1 protein. Our results suggest that spermine induces the release of spermine-released proteins by changing DNA conformation. Binding of these proteins might be sensitive to long-range changes in chromosome structure caused by torsional stress. 相似文献
14.
LIRUNSHENG JIAKETSO 《Cell research》1992,2(2):103-117
Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles. 相似文献
15.
S Levasseur L Henricks T Poleck Y Friedman G Burke 《Journal of cellular biochemistry》1985,28(4):299-306
Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic-AMP-dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6-2.4-fold increase in cytosolic cyclic-AMP-independent (messenger-independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger-independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger-independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid-dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin-regulated ornithine decarboxylase activity in thyroid. 相似文献
16.
M A de las Heras S I Gonzalez R S Calandra 《Journal of reproduction and fertility》1992,96(1):323-330
Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens. 相似文献
17.
Protein phosphorylation in chick kidney. Response to parathyroid hormone, cyclic AMP, calcium, and phosphatidylserine 总被引:1,自引:0,他引:1
The regulation of endogenous protein phosphorylation by parathyroid hormone (PTH) was investigated using confluent monolayer cultures of chick kidney cells. Homogenates and subcellular fractions of PTH (bovine 1-34)-treated cells were subjected to an endogenous protein phosphorylation assay using ((gamma- 32P]ATP in the presence or absence of 2.0 microM cAMP or 0.5 mM Ca2+ with 25 micrograms/ml of phosphatidylserine and reactions terminated with sodium dodecyl sulfate. In other experiments, cultures were incubated in a phosphate-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline containing 50 muCi/ml of [32P]PO4 and incubations were terminated with sodium dodecyl sulfate. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP stimulated 32P incorporation into proteins having molecular weights of 17,000, 22,000, 35,000, 42,000, 54,000, 75,000, 80,000, 120,000, and 143,000. Calcium-phosphatidylserine stimulated the phosphorylation of proteins of 20,000, 52,000, 58,000, 60,000, and 143,000. The protein phosphorylation patterns in cultured kidney cells and freshly isolated kidney tissue were quite similar. Treatment of cultured cells with 5-50 ng/ml of PTH resulted in stimulated phosphorylation of the 35,000 and 42,000 dalton proteins as assessed by endogenous phosphorylation in homogenates. In intact cells incubated with [32P]PO4, PTH stimulated most noticeably the phosphorylation of the 35,000-dalton protein. Based on studies with cultured and fresh kidney cells, the majority of the substrate proteins for cAMP and calcium-dependent protein kinases were located in the cytoplasm with the exception of the 42,000-dalton protein which was located in the brush-border-plasma membrane fraction. The cytoplasmic cAMP-dependent protein kinase activity was responsible for the majority of PTH-stimulated protein phosphorylation. 相似文献
18.
The binding and phospholipase A2 activity of an 11,000-dalton beta-bungarotoxin, isolated from Bungarus multicincutus venom, have been characterized using rat brain subcellular fractions as substrates. 125I-labeled beta-bungarotoxin binds rapidly (k = 0.14 min-1 and 0.11 min-1), saturably (Vmax = 130.1 +/- 5.0 fmoles/mg and 128.2 +/- 7.1) fmoles/mg), and with high affinity (apparent Kd = 0.8 +/- 0.1 nM and 0.7 +/- 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynpatic membranes. The 11,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine greater than phosphatidyl choline greater than phosphatidyl serine. These results indicate that the 11,000-dalton beta-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 11,000-dalton toxin are contrasted with those of the beta-bungarotoxin found in highest concentration in the venom (the 22,000-dalton beta-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that beta-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynatic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability. 相似文献
19.
When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease. 相似文献
20.
Yuan Q Ray RM Viar MJ Johnson LR 《American journal of physiology. Gastrointestinal and liver physiology》2001,280(1):G130-G138
Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein. 相似文献