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.     

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 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.     

Formation of creatine phosphate from creatine and 32P-labelled ATP by isolated rabbit heart mitochondria

With ATP [γ-³²P] we have demonstrated directly that mitochondrial creatine phosphokinase catalyzes the formation of large amounts of creatine phosphate with mitochondria generated ATP as substrate rather than added extramitochondrial ATP.     

31P and 17O NMR of Mn(III)-containing acid phosphatase: Evidence for direct metal-phosphate interaction and oxygen exchange from water into inorganic phosphate

The acid phosphatase isolated from sweet potato tubers by us is unique Mn(III)-containing enzyme which hydrolyzes phosphomonoesters and nucleotide phosphates. The present ³¹P and ¹⁷O NMR studies of the Mn(III)-containing acid phosphatase solved two important problems. The broadening of the phosphate ³¹P resonance signal in the 1:1 enzyme-substrate system shows evidence for direct metal-phosphate interaction in the Mn(III)-containing acid phosphatase. In addition, the ¹⁷O NMR evidence for oxygen exchange from water into inorganic phosphate strongly indicates that the Mn(III)-containing acid phosphatase catalyzes an apparent transition state displacement and P-O cleavage as follows: $\text{ROPO}{}_3{}^{\mathrm{=}}\text{+ H}{}^{17}\text{OHROH + H}{}^{17}\text{OPO}{}_3{}^{\mathrm{=}}$.     

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.     

31P NMR of DNA in eukaryotic chromosomal complexes

DNA complexed with histones in soluble chromatin and with protamines in the heads of sperm has been studied by ³¹P NMR spectroscopy. Because of the large size of these nucleoprotein structures, methods of high resolution solid state NMR were employed. Proton decoupled ³¹P NMR spectra of these complexes in solution yield anisotropic chemical shift powder patterns, which indicate that the DNA is substantially immobilized by interactions with the proteins. Rapid rotation of these samples at the magic angle gives single line spectra with an isotropic chemical shift indistinguishable from DNA in the absence of proteins or that in mononucleosome core particles; this argues that packaging of the DNA by the proteins does not introduce major distortions in a predominant fraction of the phosphodiester linkages present.     

Effect of different soil and nitrogen fertilizer conditions on 32P-labelled phosphate uptake by two grass species

In two years of trials, roots of ryegrass took up more ³²P-labelled phosphate than roots of fescue. Application of 672 kg N ha^-1 increased phosphate absorption compared with application of 112 kg N ha^-1. Roots in mineral soil absorbed more phosphate than those in peat soil. In both soils uptake decreased as depth of phosphate injection increased from 5 to 30 cm. An interaction occurred whereby roots in the intermediate depth (10–22-5 cm) in peat absorbed less phosphate than in mineral soil and this was apparently unrelated to the exchange or sorption properties of the soil.     

Varietal differences in uptake of 32P labelled phosphate in clover plus ryegrass swards and monocultures

Lolium perenne cv. S.23, L. multiflorum cv. RvP, and Trifolium repens cvs S.184 and Olwen, were grown in mixed sward and monoculture during 1979. Whereas in mixtures grass roots absorbed more ³²P than clover roots, in monoculture clover generally absorbed more ³²P than grass roots. This showed that grass was a very strong competitor for uptake in mixed swards. Clover and grass monocultures absorbed most ³²P from 10 or 15 cm depth in the soil, while grass in mixtures absorbed most ³²P at 22.5 cm depth. Comparing varieties, in monocultures in June, Olwen was most active in absorbing ³²P at 15 cm. In August, Olwen absorbed more at 15 cm and 22.5 cm than S.184 or the grass varieties. Differences in absorption depth between varieties were less in mixtures than in monocultures. S.23 absorbed more ³²P in the late season than RvP, both in monoculture and in mixtures. Thus Olwen differed from S. 184 in depth and timing of uptake, whilst S.23 differed from RvP in time of uptake. Such varietal differences could be exploited by manipulation of depth and timing of fertiliser application to increase the precision of sward management.     

An exchange enzyme-catalyzing incorporation of inorganic phosphate into adenosine diphosphate in Alcaligenes faecalis

The reaction in the Alcaligenes faecalis system previously believed to be a stoichiometric ATP-forming process catalyzed by a high-energy intermediate of oxidative phosphorylation has been shown to be a catalytic ADP ? P_i exchange reaction, which incorporated P_i into the β position of ADP. Preincubation of the enzyme with P_i was partially inhibitory (10–20%), and this substrate-preconditioning effect was greatly enhanced (up to 80%) by divalent cations. DEAE-Sephadex profiles showed several peaks of activity while sucrose-gradient profiles showed only one. About 95% of the enzyme was soluble, while the remainder was compartmentalized in the membrane fraction; most of this small amount could be leached out slowly by successive washings. Low ionic strength reduced but did not eliminate the compartmentation. Incubating phosphorylating particles with DPNH resulted in a slight increase in the elution of coupling factors and exchange enzyme over that of the control system. This was explained in terms of small effects on the affinity of coupling factors for membranes and on the degree of compartmentation of the exchange enzyme. The identity of the enzyme was established as polynucleotide phosphorylase by the finding that it could form a trichloracetic acid-precipitable product which was RNase sensitive (ribopolynucleotide) from [³H] ADP. The concentration of divalent cations determined whether the enzyme would catalyze primarily ADP ? P_i exchange or synthesize polynucleotide.     

A membrane component of the cellular slime mouldDictyostelium discoideum rapidly labelled with [32P]orthophosphate

After labellingDictyostelium discoideum (Strain Ax-2) for 30 min with [³²P]-orthophosphate a material was observed in cytoplasmic extracts, which cosedimented with polyribosomes in sucrose density gradients. The radioactivity in this material was insensitive to ribonuclease and deoxyribonuclease but was partially solubilized by alkali. All the radioactivity was rendered soluble in trichloroacetic acid by treatment with sodium deoxycholate. Most of the³²P counts were extractable in chloroform-methanol (2:1, v/v) and upon analysis by thin-layer chromatography the isotope was found in various phospholipids, chiefly phosphatidylethanolamine with some in lecithin and phosphatidylserine. Upon examination in an electron microscope the material was found to be composed of membrane, glycogen and an unidentified amorphous material.     

Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins

A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of ¹⁴C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the ¹⁴C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the ¹⁴C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the ¹⁴C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.     

The interaction between water and the polar head in inverted phosphatidylcholine micelles A 2H and 31P relaxation study

²H and ³¹P spin-lattice relaxation times (T₁) were studied for invented egg phosphatidylcholine micelles in CCl₄ as functions of ²H₂O concentration. When the ²H₂O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T₁ of ³¹P increased by about 2.6 fold, whereas T₁ of ²H increased by about 50 fold. A quantitative analysis of the deuterium T₁ data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation energy for the deuterium T₁ was 7.1 ± 0.8 kcal/mol (30 ± 3 kJ/mol), and was independent of the ²H₂O concentration.     

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.