Phosphorylation of membrane-located proteins of soybean in vitro and response to auxin

Isolated membranes of soybean incorporate ³²P from γ-[³²P]ATP in vitro. The incorporation was rapid and did not require added calcium. When displayed on 10% sodium dodecyl sulfate-polyacrylamide gels, several protein bands were revealed. An apparent auxin (2,4-dichlorophenoxyacetic acid) stimulation of ³²P incorporation into material from membrane vesicles insoluble in trichloroacetic acid-perchloric acid may be reflected partly in enhanced incorporation into protein bands with apparent molecular weights of 45,000 and 50,000. Additionally, a low molecular weight component was sometimes observed where incorporation was stimulated 2- to 3-fold by auxin. However, protein-bound radioactivity represented only a small fraction of the total radioactivity of the acid-insoluble material. Other labeled constituents, not retained on the gels, may contribute to the apparent, rapid (10 s or less) auxin response of the isolated membranes. Stimulation of incorporation into the low molecular weight component was given by diglyceride plus calcium, constituents known to augment protein kinase activities in other systems.     

The fate of exotoxin of Bacillus thuringiensis in Galleria mellonella caterpillars

Three hours after parenteral administration of ³²P-labeled exotoxin of Bacillus thuringiensis to caterpillars of Galleria mellonella, 80% of the radioactivity was localized in the hemolymph in the form of the original exotoxin. The remaining radioactivity occurred in the organs of the caterpillar, especially in the spinning glands and the intestine. After peroral administration, the exotoxin does not pass the intestinal wall into the hemolymph to a measurable degree. In this case, the exotoxin is split in the intestinal wall and the products of ³²P reutilization have been found in the hemolymph. The mechanism of action of the exotoxin in the insect organism is discussed; presumably it depends on different ways of administration of the substance.     

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme a reductase: Analysis of 32P-labeled,inactivated enzyme

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg²⁺ and [γ-³²P]ATP, then solubilized and purified to homogeneity. The ³²P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, ³²P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg²⁺ and ATP.     

Distributional changes of 32P-labeled acid-soluble phosphates and phospholipids among subcellular fractions during early vertebrate embryonic development

Early developing embryos of the toad Bufo arenarum Hensel were employed to study the content and in vivo labeling with ³²P of the acid-soluble phosphates and phospholipids at the subcellular level. The radionuclide was administered to the female toad along with the pituitary extract used to induce the ovulation.Most of the total phospholipids (68%) and proteins (84%) are confined to the yolk platelet fractions. Up to the heart beat stage (130 h of development) there are no significant changes detectable in protein and phospholipid content.The total P content in trichloroacetic acid-soluble fraction was distributed mainly between postmitochondrial supernatant (58%) and yolk platelet fraction (37%) in the unfertilized oocyte. As development proceeds an increase was observed in the former and a decrease in the latter. The acid-solube phosphates in the mitochondrial fraction only amount to 4% of the total embryo throughout the examined stages.The unfertilized oocyte contains about 98% of acid-soluble phosphates labeled with ³²P in the postmitochondrial supernatant and as development proceeds a striking decrease was found to occur while the radioactivity in the acid-soluble phosphates of mitochondrial and yolk platelet fractions increases significantly during the studied stages. About 11.5% of the lost radioactivity from the acid-soluble phosphates was found to be used to label the phospholipids.     

A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart

(i) A new, rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts in the presence of other ³²P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of ³²P into protein from [γ-³²P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) ³²P_i uptake in perfused rat heart was found to be a relatively slow process, with a K_m of 0.084 mm, whereas equilibration between intracellular ³²P_i and [γ-³²P]ATP occurred rapidly.     

Convenient methods to determine the specific radioactivity of (gamma-32P)ATP of high specific activity.

A satisfactory method for the determination of the specific activity of highly labeled [γ-³²P]ATP has not been reported previously. Yields of high specific activity ³²P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to ³H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled ³²P material since the crossover into the ³H channel will greatly exceed the radioactivity of the ³H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-³²P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-³²P]ATP. This procedure measures the phosphate transfer from [γ-³²P]ATP to oligothymidylic acid [dT(pT)₁₀] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-³²pT(pT)₁₀, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined.     

Transformations between organic and inorganic sediment phosphorus in Lake Balaton

In order to estimate microbial P content and biological P uptake in sediments, the tungstate precipitation method of Orrett & Karl (1987) was used in sediment extracts. This method allows a simple and rapid separation of organic and inorganic ³²P radioactivity. Either inorganic ³²P (as carrierfree H₃ ³²PO₄) or organic ³²P (as ³²P-labelled algal material) was added to surface sediment suspensions of shallow Lake Balaton. Inorganic ³²P was rapidly transformed into organic ³²P, and this process was completely inhibited by formaline. P content of living benthic microorganisms was estimated from steady state distribution of the radioactivity. Transformation of algal organic P into inorganic P could also be detected.In extremely P limited Lake Balaton benthic microorganisms were shown to supplement their high P requirements by inorganic P uptake. The velocity of the inorganic into organic P transformation, i.e. the rate of microbial P uptake, was comparable to P uptake in the water column. Microbial P uptake contributed significantly to total P fixation by sediments, particularly at low ( 100 µg P l^–1) phosphate additions.     

Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XII. Evidence for interaction of the acyl phosphate at the active site with neighboring groups

The sodium-potassium adenosinetriphosphatase (NaK ATPase), partially purified from beef brain, has been phosphorylated with [γ-³²P]ATP in the presence of Na and Mg and digested with pronase. A single ³²P-labeled peptide spot has been identified on paper electrophoresis, accounting for 60% of the radioactivity in the ³²P-labeled enzyme, the remainder of the radioactivity being [³²P]-orthophosphate resulting from breakdown of the highly labile acyl phosphate during pronase digestion. The ³²P in the pronase peptide was released as [³²P]-orthophosphate by N-propylhydroxylamine—as to be expected of an acyl phosphate compound. The pH stability of the acyl phosphate in the denatured phosphorylated NaK ATPase, in the pronase peptide and in acetyl phosphate were quite different. The phosphorylated protein had the lowest stability of higher pHs, acetyl phosphate had the highest stability, and the pronase peptide had an intermediate stability. These results indicate that the neighboring groups in the polypeptide chain containing the acyl phosphate residue influence the stability of the acyl phosphate bond.     

Lack of phosphate incorporation into TN-I in live frog muscle

The phosphorylation of TN-I was investigated in muscles of live frogs injected with [³²P]orthophosphate. Isolation of TN-I was carried out by the rapid and specific technique of affinity chromatography developed by Syska, Perry, and Trayer [FEBS Lett.40, 253–257 (1974)] followed by gel electrophoresis in the presence of sodium dodecyl sulfate. No significant labeling of TN-I was found even in frogs which were exposed to the ³²P-treatment for several days. A comparison of the specific radioactivity of TN-I from resting and contracting frog muscle showed no change in the ³²P content of TN-I during muscle contraction.     

Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

Autoradiography: A sensitive method in dispersal studies with Adoxophyes orana (Lepidoptera: Tortricidae)

By mixing ²²Na and ³²P in artificial diets the adults of A. orana and their subsequent stages were labelled to study their dispersal. The sensitivity of a Geiger-Müller tube and of autoradiography were compared. The latter method was about 70 times more sensitive under our experimental conditions and was therefore preferredto detect the radioactivity in the insects recaptured during the release experiments. As well as the great sensitivity to beta rays of all energies, autoradiography has the advantage that it can be used where little scientific equipment is available. ³²P was a suitable source of radioactivity. The radioactivity of spermatophores and eggclusters of parents labelled with it could be demonstrated easily. In spite of the short half life of ³²P, it was possible to detect radioactivity in a spermatophore extracted from a second generation female, even when modest doses of isotope (200 Ci/800 ml diet) had been used to label the first generation.Field experiments showed that isotopes could be used to show the distance of primary and secondary dispersal of A. orana.

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Zusammenfassung Zur Untersuchung ihrer Ausbreitung wurden erwachsene Adoxophyes orana und ihre Folgegeneration durch Beimischung von ²²Na und ³²P zu künstlichen Diäten der Raupen markiert. Die Empfindlichkeit eines Geiger-Müller-Zählers und der Autoradiographie wurden verglichen. Die letztgenannte Methode erwies sich unter unseren Versuchsbedingungen als 70mal sensitiver und wurde deshalb zum Nachweis der Radioaktivität von Insekten bevorzugt, die bei Freilassungsexperimenten wiedergefangen wurden. Neben der großen Empfindlichkeit gegenüber -Strahlen aller Energiebereiche hat die Autoradiographie den Vorteil, daß sie auch dort benutzt werden kann, wo nur geringe wissenschaftliche Ausrüstung verfügbar ist. ³²P war dafür geeignetes radioaktives Material. Die Radioaktivität der Spermatophoren und Eigelege der mit ³²P markierten Falter konnte leicht nachgewiesen werden. Trotz der kurzen Halbwertszeit von ³²P war es möglich, die Radioaktivität in einer Spermatophore nachzuweisen, die aus einem Weibchen der zweiten Generation entnommen wurde, selbst wenn nur mäßige Dosen des Isotops (200 Ci/800 ml Diät) benutzt worden waren, um die erste Generation zu markieren.Feldversuche zeigten, daß die Isotope dafür benutzt werden können, die Entfernung der primären und sekundären Dispersion von A. orana nachzuweisen.

     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

Studies on the methods of inorganic nutrient application in coconut

Summary Using carrier free³²P, tagged single superphosphate and⁸⁶Rb, the efficiency of different methods of plant injection and soil placement techniques for fertilizer application was examined. In the plant injection techniques the radioactivity was fed to the palms through growing roots tips, cut ends of roots, stem injection and leaf axils. The application of radioactivity through the cut ends of roots was most efficient since³²P was detected in 10 m tall palms, four hours after application. In stem, leaf axil and growing roots tips injection the³²P was detected after 8, 12 and 18 h. Out of four methods of soil application, the quickest recovery of³²P in the palms was detected after 7 days of placement when applied by the hole method. The³²P activity in the palms through circular trenches, strips and basin methods was recorded after 8, 8 and 11 days of application respectively. The accumulation of⁸⁶Rb was significantly higher than³²P. With plant injection technique the accumulation of activity was found to be significantly higher than with soil placement methods. The rate of radioactivity absorption was 10 to 60 time faster in the former technique as compared to that of the latter. The application of radioactivity through cut ends of roots and circular trench methods, were found to be better and may be recommended for nutrient application in coconut.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

RAPID AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG OF FUCOSE-, GLUCOSAMINE- AND SULPHATE-CONTAINING MATERIAL

—An in vitro system from the frog has been used to study fast axonal transport of glycoproteins. The migration of [³H]fucose-, [³H]glucosamine- and [³⁵S]sulphate-labelled material was followed from the dorsal ganglia, along the sciatic nerve towards the gastrocnemius muscle. The distribution in different subcellular fractions, effect of cycloheximide and transport kinetics did not differ very much between fucose- and glucosamine-incorporation into the nerve. Cycloheximide blocked the synthesis of TCA-insoluble radioactivity, which was transported at a rate of 60–90 mm per day at 18°C, more effectively than the synthesis of stationary proteins in the ganglia. About 10 per cent of the TCA-insoluble and transported radioactivity was extracted by chloroform-methanol (2:1, v/v) and might be glycolipids and the rest glycoproteins. Results suggest that TCA-soluble activity, which was recovered in the nerve, originated in part from labelled macromolecules consumed along the axons. The rapidly transported TCA-insoluble radioactivity was 85 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. [³⁵S]Sulphate-labelled TCA-insoluble material was resistant towards chloroform-methanol (2:1, v/v) extraction and rapidly transported from the ganglia into the nerve. The synthesis was inhibited by cycloheximide. The material, probably proteoglycans, represented a quantitatively minor part of transported glycoproteins.     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 μM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [γ-³²P]ATP and [γ-³²P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [γ-³²P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [γ-³²P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.