The incorporation of P into spectrin aggregates following incubation of erythrocytes in P-labelled inorganic phosphate

³²P was incorporated into spectrin by incubation of fresh erythrocytes with ³²P_i and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appears not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.     

The incorporation of 32 P into spectrin aggregates following incubation of erythrocytes in 32 P-labelled inorganic phosphate

32P was incorporated into spectrin by incubation of fresh erythrocytes with 32Pi and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appers not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.     

Human erythrocyte spectrin: Phosphorylation in intact cells and purification of the 32P-labeled protein in a non-aggregated state

[³²P]spectrin (0.5 Ci/mMole) has been isolated from human erythrocytes incubated with ³²Pi and purified to homogeneity by preparative rate zonal sedimentation on linear sucrose gradients. ³²P-label, localized in band 2, co-elutes with spectrin from ghosts with a similar dependence on ionic strength and Mg⁺⁺ ion, and has the same sedimentation coefficient and an identical effective Stokes radius. [³²P]spectrin reassociates in a specific manner with spectrin-depleted membranes. Bands 1 and 2 bind in equal ratios, and the ³²P-label is distributed with band 2. Purified [³²P]spectrin is not aggregated since this protein migrates as a symmetrical peak on Sepharose(C1)4B at about 1.6 Vo and sediments at 8S_20,w on sucrose gradients.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

The relationship between the incorporation of 32P into phosphatidic acid and phosphatidylinositol in rat parotid acinar cells

Muscarinic and α-adrenergic stimulation of rat parotid acinar cells increases the turnover of phosphatidylinositol and phosphatidic acid. It is thought that this is initiated by hydrolysis of phosphatidylinositol, which would predict an increase in ³²P incorporation into phosphatidic acid before phosphatidylinositol. We have demonstrated an increase in ³²P incorporation into the former within 1 minute and into the latter by 2 minutes. The initial rapid rate of ³²P incorporation into phosphatidic acid slows, and the ³²P content reaches a steady state after 15 minutes. During the first 2 minutes after the addition of atropine to carbamylcholine stimulated cells, ³²P is lost from phosphatidic acid, and an equal amount is gained by phosphatidylinositol, after which ³²P incorporation equals that of the control. In cells prelabelled with ³²P, carbamylcholine, in the presence of oligomycin stimulated the loss of ³²P from phosphatidylinositol but had no effect on phosphatidic acid.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pK_a value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.     

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane

Ghosts derived from bovine chromaffin granules have a ³²P_i-ATP exchange activity which is associated with the H⁺ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (K_m for ATP and P_i, ATP/Mg²⁺ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The ³²P_i-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a ³²P_i-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F₁-ATPase were major components of the preparation.     

Deuterium and carbon-13 tracer studies of ethanol metabolism in the rat by 2H, 1H-decoupled 13C nuclear magnetic resonance

[1-¹³C, 1,1-²H₂] ethanol and [2,2,2-²H₃] ethanol were administered to bile fistula rats. A new technique, ²H, ¹H-decoupled ¹³C nuclear magnetic resonance, was used in attempting to account for the distribution of the isotopic species along the steroid skeleton of 3–45 mg of isolated bile acids. The technique revealed ²H incorporation at many carbon sites unambiguously, but has limitations as a quantitative ²H assay at these levels of sample availability.     

Shewanella putrefaciens CN32对粘土附着态铵氮释放的影响

【目的】研究产胞外分泌物微生物Shewanella putrefaciens CN32对土壤中常见粘土矿物附着态NH_4~+的释放效果及影响机制。【方法】以吸附NH_4~+的蒙脱石、蛭石、伊蒙混层矿物和黑云母为对象,通过监测S. putrefaciens CN32作用下不同粘土释放的NH_4~+含量及过程,以及监测微生物量及释放的胞外聚合物(Extracellular Polymeric Substances,EPS)的含量变化,研究S. putrefaciens CN32作用下不同粘土矿物类型附着态NH_4~+释放的差异性。【结果】粘土矿物附着态NH_4~+含量从高到低依次为蒙脱石蛭石伊蒙混层矿物黑云母(黑云母NH_4~+吸附量极低,会在非生物作用下几乎完全释放),CN32作用下粘土附着态NH_4~+相对释放量依次为蒙脱石伊蒙混层矿物蛭石;然而,尽管CN32有效促进了粘土附着态NH_4~+释放,但释放的NH_4~+并未在溶液中大量累积,而是多被微生物同化吸收转化为生物有机氮(EPS为主)并吸附在粘土表面,且粘土对EPS的吸附能力表现为蒙脱石伊蒙混层矿物蛭石黑云母;由于粘土吸附NH_4~+及EPS都与矿物中的羟基(结构水或层间水)关系密切,推测EPS对矿物羟基的竞争吸附可能是CN32促进NH_4~+释放的重要原因之一。【结论】以上结果表明,产EPS微生物S. putrefaciens CN32能够促进各类粘土矿物的附着态NH_4~+释放,但释放的NH_4~+可以通过微生物作用转化为有机氮,从而在减少NH_4~+流失的同时增加土壤氮肥的生物可利用性,因此微生物在降低土壤氮肥流失、转化土壤氮肥污染过程中可能起到了重要作用,也揭示了深入系统地分析不同类型土壤(粘土类型不同)中粘土附着态NH_4~+在不同功能微生物作用下的迁移转化过程,是精准评估土壤氮肥施用效率及流失风险的前提之一。     

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

In the present study, we examined the effects of long- and short-term hypoxia on net uptake and transport of phosphorus to shoots of pond pine (Pinus serotina Michx.), a moderately flood-tolerant southern pine, and the influence aerenchyma formation might have in maintenance of P uptake and transport. Seedlings were grown under aerobic (250 μM O₂) or hypoxic (≤50 μM O₂) solution conditions for 5.3 weeks in continuously flowing solution culture containing 100 μM P. Intact seedlings were then labeled with ³²P for up to 24 h to determine how short- and long-term hypoxic solution conditions affected rates of unidirectional influx and the accumulation of ³²P in roots and shoots. Seedlings in the long-term hypoxic treatment were grown for 5.3 weeks in hypoxic solution and also labeled in hypoxic uptake solution. The short-term hypoxic treatments included a 24-h hypoxic pretreatment followed by time in labeled hypoxic uptake solution for seedlings grown under aerobic or hypoxic conditions; in the latter case, diffusion of atmospheric O₂ entry into stem and root collar lenticels was blocked, thus removing any influence that aerenchyma formation might have had on enhancing O₂ concentrations of root tissue. Although unidirectional influx rates of ³²P in roots of seedlings grown under long-term hypoxic conditions were 1.4 times those of aerobically grown seedlings, accumulation of ³²P in roots was similar after 24 h in labeled uptake solution. These results suggest that ³²P efflux was also higher under hypoxic conditions. Higher shoot/root fresh weight ratios and lower shoot P concentrations in seedlings grown under hypoxic solution conditions suggest that the “shoot P demand” per unit root should be high. Yet accumulation of ³²P in shoots was reduced by 50% after 24 h in hypoxic uptake solution. Both short-term hypoxic treatments decreased accumulation of ³²P in roots by more than 50%. Short-term hypoxia decreased shoot accumulation in seedlings grown under aerobic and hypoxic conditions by 84 and 50%. respectively. Short- and long-term hypoxic conditions increased the percentage of root ³²P in the nucleic acid and chelated-P pools, resulting in a significantly smaller percentage of ³²P in the soluble inorganic phosphate (p_i) pool, the pool available for transport to the shoot. However, a reduction in pool size or in labeling of the pool available for transport cannot fully account for the large reduction in accumulation of ³²P in shoots, particularly in the short-term hypoxic treatment of aerobically grown seedlings. Our results suggest that both influx and transport of ³²P to shoots of pond pine seedlings are O₂-dependent processes, and that the transport of ³²P to shoots may be more sensitive to hypoxic solution conditions than influx at the cortical and epidermal plasmalemma, with aerenchyma formation supporting a substantial amount of both ³²P uptake and transport.     

Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled,tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little ³²P_i, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [³H]uridine before the experiment provides a measure of ribosome yield. The amount of ³²P_i incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [³²P]ATP increases, when the cells are stimulated by prostaglandin F_2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the ³²P_i incorporated into S6 increases by a factor greater than the increase in the specific activity of [³²P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.     

DDT EFFECT ON 32P INCORPORATION FROM γ-LABELLED ATP INTO PROTEINS FROM LOBSTER NERVE

The insecticide DDT (1,1,1 trichloro-2,2-bis-(p-chlorophenyl) ethane) was found to affect the amount of ³²P from [γ-³²P] ATP incorporated into proteins derived from lobster peripheral nerves in a variety of ways depending upon the relative concentrations of ATP, Na⁺, K⁺, Mg²⁺, and Ca²⁺. When a high concentration of ATP (2.5 ± 10^?5m ) was used DDT (10^?5m ) inhibited the incorporation and reversed the increased incorporation caused by ouabain. At low concentration of ATP (8.6 ± 10 ^?8m ), DDT inhibited the incorporation when the buffer contained Mg²⁺ and Na⁺ or K⁺ alone, but when the buffer contained Mg²⁺ and both Na⁺ and K⁺ together, DDT consistently caused an increased amount of ³²P to be incorporated. The ability of DDT to cause increases in incorporation of ³²P into the lobster nerve proteins was found to parallel many of the properties that this compound shows when allowed to poison crustacean nerves. For example: the effect was negatively temperature dependent, Ca²⁺ lowered the incorporation in the presence of DDT, and DDT consistently caused greater amounts of ³²P to be incorporated than its less potent analog DDE (1,1-dichloro-2,2-bis-(p-chlorophenyl)ethylene). The proteins that were affected by DDT were microsomal in nature and could be centrifuged from supernatant by recentrifuging at 149,000 g. The possibility that this system may be the actual target through which DDT causes its characteristic interferences of the ionic conductance changes associated with the action potential are discussed.     

Spectrum of 32P-induced mutants of Caenorbabditis elegans

Mutants of the free living nematode C. elegans were isolated by using ³²P as a mutagen. It is shown that most of these mutants arise from ³²P suicide. A comparison of EMS-induced and ³²P-induced autosomal recesive mutations shows that there are no large regions of C. elegans genome which are protected from chemical mutagenic action of EMS.     

The synthesis of unlabeled and 32P-labeled phosphocitrate and analytical systems for its identification

A method for the synthesis of phosphocitrate is described using 2-cyanoethyl phosphate to phosphorylate triethyl citrate. Following alkaline hydrolysis of the coupled intermediate, phosphocitrate was purified by ion-exchange chromatography on an AG 1-X8 (HCO₃^?) column. The method was also used to prepare [³²P]phosphocitrate. Phosphocitrate was characterized by ¹H NMR, ³¹P NMR, and ¹³C NMR spectroscopy. In addition methods for thin-layer chromatography and enzyme assay are detailed for the detection of phosphocitrate.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with $\text{p-}\text{mercuribenzene sulphonate}$ (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with $\text{N-}\text{ethylmaleimide}$ to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.     

The enzymic synthesis of β-[32P]UDP-N-acetylglucosamine

Cells of Micrococcus sp. 2102 incorporate inorganic [³²P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[³²P]phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP: N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise β-[³²P]UDP-N-acetylglucosamine.     

Diapause hormone action in silkworm ovaries incubated in vitro; 14C-trehalose incorporation into glycogen

Developing ovaries from pharate adults of the silkworm, Bombyx mori, were incubated in a medium containing ¹⁴C-trehalose or ¹⁴C-glucose, and the effects of diapause hormone on the incorporation of these isotopes into ovary glycogen were studied. The rates of incorporation of ¹⁴C-trehalose remained unchanged in ovaries incubated for 36 hr when the medium was renewed at intervals of 12 hr, and showed saturation kinetics against the concentration of the sugar in the medium, giving apparent K_m values of 6·0 mM for trehalose.There was no difference in ¹⁴C-glucose incorporation by ovaries grown in the presence (+SG) and absence (?SG) of the suboesophageal ganglion (SG). However, when ¹⁴C-trehalose was used as a substrate for glycogen synthesis, there was a marked difference in the incorporation between them, i.e. the incorporation was more than 60 per cent higher in +SG ovaries than that in ?SG ovaries. Increased incorporation of ¹⁴C-trehalose was also observed in ovaries from SG-removed pharate adults which received an injection of diapause hormone preparations. Maximum stimulation rates (about twofold) appeared 36 hr after the injection. Further, comparable effects on ¹⁴C-trehalose incorporation were observed in ovaries which were incubated with diapause hormone preparations added in vitro.These data are discussed in relation to the hormonal regulation of trehalase activity in ovaries.     

Studies on corneal endothelial growth and repair. II. Increased transcription as detected by incorporation of 3H-uridine and 3H-actinomycin D

Endothelial cells from injured frog corneas undergo increased ³H-uridine and ³H-actinomycin D (³H-AMD) incorporation as judged by autoradiography. The increase in ³H-AMD binding occurs when living endothelium is labeled in vitro or when fixed preparations are exposed to the drug. The changes in ³H-AMD incorporation detected by the two methods are comparable (55 and 62 % for living and pre-fixed tissue respectively). However, when fixed endothelium is also de-histonized with 2 N HCl, differential binding of ³H-AMD is eliminated. This result suggests that the enhanced incorporation of ³H-AMD into nuclei is at least partly due to a modification in the association of chromosomal proteins with DNA and not entirely to cell permeability changes that may accompany wound repair. This contrasts with observations of cells that are killed outright by the injury. Such cells bind very large amounts of ³H-AMD compared with their living neighbors. Here the difference in incorporation is eliminated by prefixation. Thus, in the dead cells increased binding may be due to a reduction of cell surface permeability barriers which accompanies cell morbidity.