Distribution Of 32P Research Articles

Impact of Mycorrhizae Formation on the Phosphorus and Heavy-Metal Uptake of Alfalfa

Three pot experiments were set up to determine how efficiently mycorrhizal fungi affect the uptake, translocation, and distribution of labeled phosphorus (32P), phosphorus (P), and heavy metals in alfalfa (Medicago sativa L.). In experiments 1 and 2, the efficiencies of different arbuscular mycorrhizal fungi (AMF) species including Glomus mosseae, G. etunicatum, G. intraradices and a mixed strain (G. mosseae, Gigaspora hartiga, and G. fasciculatum) on uptake, translocation, and distribution of 32P and P in alfalfa were investigated, respectively. In a third experiment, the efficiency of G. mosseae on uptake and distribution of heavy metals [cadmium (Cd), cobalt (Co), lead (Pb), and combinations] was tested. Results of experiments 1 and 2 suggest that G. mosseae was the most effective at increasing the uptake of 32P and P. Experiment 3 result showed that in the triple-metal-contaminated soil, inoculated plants had greater Co (32.56 mg kg−1) and Pb (289.50 mg kg−1) concentration and G. mosseae enhanced the translocation of heavy metals to shoot. Hence, mycorrhizal alfalfa in symbiosis with G. mosseae can be used for remediation of heavy metals polluted soils with high efficiency.

A Facile, Viable Approach Toward the Preparation of 32P Patches for the Treatment of Skin Cancer

A facile, viable, "green" two-step, inexpensive technique was developed for the preparation of (32)P patch for the treatment of skin cancer. This technique consists of impregnation of H(3)(32)PO(4) solution into an inert solid carrier followed by immobilization into a nonleachable matrix by lamination. The morphology of the impregnated paper was evaluated by scanning electron microscope and energy-dispersive spectral analyses. Radioactive patches containing up to ∼37 MBq/cm(2) of (32)P could be prepared. Distribution of (32)P on sources was uniform and release of (32)P from the sealed source in water and saline was found to be well within the permissible levels of 185 Bq. Custom-shaped (32)P-patches after quality assurance were supplied to AIIMS, New Delhi, for clinical evaluation. (32)P-impregnated paper protected by a laminated film holds promise for treatment of superficial cancers.

32P-chromic phosphate-poly (L-lactide) implantation brachytherapy on prostatic carcinoma with lymphatic metastasis

Objective To study the anticancer effects of 32P-chromic phosphate-poly ( L-lactide) (32P-CP-PLI^A) in nude mice with lymphatic metastasis of prostatic carcinoma after implantation brachytherapy. Methods The nude mice were intratumorally implanted with 32P-CP-PLLA of 3.7, 7.4, 14.8 MBq after prostatic carcinoma solid tumor model was established for four weeks. The distribution of 32P in body was observed. The morphology of the tumor and regional lymph nodes was examined. White blood cells and platelets were counted in peripheral blood. Results 32P mainly remained in the tumor and regional lymph nodes. Morphological examination demonstrated that the solid tumor foci necrosed thoroughly, normal structure of regional lymph nodes disappeared, which was replaced by necrotic tissue and hemorrhage. The tumor growth inhibition rate was positively correlated to administration doses. When the doses were 3.7, 7.4, 14.8 MBq, the tumor growth inhibition rate was (70.16 ±5.48)% , (80.18 ±5.84)% , (84.97 ±4.79)% , respectively. No obvious bone marrow depressions were detected. Conclusion The intratumoral implantation with 32P-CP-PLLA can produce anticancer effects on the solid tumor foci and regional lymph nodes. Key words: 32P-chromic phosphate; Prostatic carcinoma; Lymphatic metastasis; Implantation brachytherapy

Dose‐rate distribution of ‐glass microspheres for intra‐arterial brachytherapy

The intra-arterial administration of radioactive glass microspheres is an alternative therapy option for treating primary hepatocellular carcinoma, the main cause of liver cancer death, and metastatic liver cancer, another important kind of cancer induced in the liver. The technique involves the administration of radioactive microspheres in the hepatic artery, which are trapped preferentially in the tumor. In this work the GEANT4 toolkit was used to calculate the radial dose-rate distributions in water from 32P-loaded glass microspheres and also from 90Y-loaded glass microspheres. To validate the toolkit for this application, the authors compared the dose-rate distribution of 32P and 90Y point sources in water with data from the International Commission on Radiation Units and Measurements report 72. Tables of radial dose-rate distributions are provided for practical use in brachytherapy planning with these microspheres. The simulations with the microspheres show that the shape of the beta ray energy spectra with respect to the 32P and 90Y sources is significantly modified by the glass matrix.

Translocation and distribution of 32P labelled potassium phosphonate in black pepper ( Piper nigrum L)

The aim of this project was to study the translocation of potassium phosphonate within black pepper ( Piper nigrum L.) and to elucidate the amount of chemical distributed through out the plant based on a tracer technique. Potassium phosphonate is a potential fungicide used against the pathogen Phytophthora capsici, which causes Phytophthora foot rot (Quick wilt) in the plant. Reports indicate that translocation of phosphonate is ambimobile, but there is no visual evidence for this. Potassium phosphonate labelled with radioactive phosphorus, 32P, was applied to the black pepper vine and bush pepper and its translocation to different parts of the pepper plant was studied using an autoradiography technique. The chemical which migrated to different parts of the plant was quantitatively estimated by measuring 32P on a liquid scintillation spectrometer following Cerenkov counting. Only traces of the chemical were lost to the soil through root systems. These results indicate that foliar sprays of potassium phosphonate to pepper could be a viable method of application for soil and foliar pathogens.

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins. Here we demonstrate that the analysis of tryptic peptides together with analysis of the full-length protein provided optimal characterization of insulin-like growth factor-binding protein-5 (IGFBP-5) phosphorylation and glycosylation. IGFBP-5 binds insulin-like growth factors with high affinity and has important roles in cell survival, differentiation, and apoptosis. Until now, the primary structure of IGFBP-5 has been incompletely defined. We analyzed human IGFBP-5 from T47D cells by mass spectrometry to determine all of the in vivo post-translational modifications. In full-length IGFBP-5, 31% of the protein was unmodified, 37% was monophosphorylated, and 4% was diphosphorylated with no other modification. The remaining 27% was glycosylated, more than half of which was also monophosphorylated. The major phosphorylation site was Ser(96) in the central domain, and a minor phosphorylation site was Ser(248) near the C terminus. Neither site was phosphorylated in vitro by casein kinase 2, ruling it out as the in vivo kinase. An in vivo phosphorylation site was also found in IGFBP-2 at an analogous position, Ser(106). IGFBP-5 was heterogeneously O-glycosylated mainly by sialylated core 1 type glycans. The most abundant structure contained N-acetylhexosamine, hexose, and two N-acetylneuraminic acid carbohydrates. A small amount of sialylated core 2 type glycan was also present. Phosphorylation and O-glycosylation both affected IGFBP-5 binding to heparin but not insulin-like growth factor binding or ternary complex formation with the acid-labile subunit. The results reveal the first description of the in vivo phosphorylation of IGFBP-5 and its glycan composition.

Radioactive sputter cathodes for 32P plasma-based ion implantation

The development of clinical treatments involving the use of beta-emitting millimetric and sub-millimetric devices has been a continuing trend in nuclear medicine. Implanted a few nanometers below the surface of endovascular implants, seeds or beads, beta-emitting radioisotopes can be used in a variety of biomedical applications. Recently, new technologies have emerged to enable the rapid and efficient activation of such devices. A pulsed, coaxial electron cyclotron resonance plasma reactor was designed and tested to demonstrate the feasibility of plasma-based radioactive ion implantation (PBRII). It has been shown that such plasma reactors allow for the implantation of radioisotopes ( 32P) into biomedical devices with higher efficiencies than those obtained with conventional ion beams. Fragments containing radioactive atoms are produced in the implanter by means of a negatively biased solid sputter cathode that is inserted into an argon plasma. Dilute orthophosphoric acid solutions (H 3 32PO 4) are used for the fabrication of flat sputter targets, since they offer a high radioisotope content. However, the aggregation of the radioactive solute into highly hygroscopic ring-like deposits rather than flat, thin radioactive films is observed on certain substrates. This article describes the effect of this nonuniform distribution of the radioisotopes on the efficiency of PBRII, and presents a technique which enables a better distribution of 32P by coating the substrates with iron. The iron coating is shown to enable optimal radioisotope sputtering rates, which are essential in 32P-PBRII for the efficient activation of millimetric biomedical devices such as stents or coils.

Resveratrol inhibits polyphosphoinositide metabolism in activated platelets

The effects of resveratrol ( trans-3,4′,5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)- P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5- P 2 ), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4- P 2 ) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5- P 3 ) were monitored in blood platelets prelabelled with [ 32P]P i. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of 32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)- P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5- P 2 available for signalling in these cells.

Phospho-imaging as a tool for visualization and noninvasive measurement of P transport dynamics in arbuscular mycorrhizas.

• A new method is described for monitoring hyphal 32 P transport in compartmented, monoxenic mycorrhizal root cultures. Nondestructive time-course measurements of P transport in hyphae were obtained by capturing digital autoradiograms on P-imaging screens, and comparing with growth observed by optical scanning. 32 P distribution measured by densitometry on the day of harvest closely agreed with values obtained by liquid scintillation counting after destructive harvest. • Virtually all labeled PO4 was absorbed by arbuscular mycorrhizal (AM) hyphae, but transfer to the roots appeared to be incomplete. P transport was not unidirectional towards the roots, as 32 P was also transported from the root compartment to the hyphal compartment. Net P flux rates were calculated for hyphae crossing between compartments, taking bidirectional flow into account. • Amounts of transported P were poorly correlated with extra-radical hyphal length and root d. wt, but highly correlated with the number of hyphae crossing the barrier separating the two compartments. Such correlations were highest when only hyphae with detectable protoplasmic streaming were considered. • The method was tested using radiolabeled P sources, H2 PO4 - and cytidine triphosphate (CTP), and the AM fungi, Glomus intraradices and G. proliferum. Fungal transport of 32 P from CTP was much slower than from PO4 for both fungi.

Effects of l-thyroxine on incorporation of 32P into phospholipids of freshwater eels

The effects of l-thyroxine on phospholipid biosynthesis, via 32P incorporation, were studied in gill, kidney, liver and muscle tissue of eels acclimatized at 11°C. l-thyroxine treatment had no effect on tissue content of lipid, inorganic and organic acid-soluble phosphorus. Only an increase of the specific radioactivities of lipid, inorganic and organic acid-soluble phosphorus was observed in the muscle. Percentage distribution of 32P among classes of phospholipid were significantly altered in liver and muscle, without change in phospholipid composition. A specific effect of l-thyroxine on 32P incorporation into phosphatidic acid in muscle and liver has been shown. As expected by the higher specific radioactivity of muscle inorganic and organic acid-soluble phosphorus, the increased incorporation of 32P into phosphatidic acid probably results from a higher specific radioactivity of muscle ATP phosphorus.

Metabolic evidence for PtdIns(4,5)P2-directed phospholipase C in permeabilized plant protoplasts.

Comparison of the sequences of the genes encoding phospholipase C (PLC) which have been cloned to date in plants with their mammalian counterparts suggests that plant PLC is similar to PLCdelta of mammalian cells. The physiological role and mechanism of activation of PLCdelta is unclear. It has recently been shown that Ins(1,4,5)P3 may not solely be the product of PtdIns(4,5)P2-directed PLC activity. Enzyme activities capable of producing Ins(1,4,5)P3 from endogenous inositol phosphates are present in Dictyostelium and also in rat liver. Significantly it has not been directly determined whether Ins(1,4,5)P3 present in higher plants is the product of a PtdIns(4, 5)P2-directed PLC activity. Therefore we have developed an experimental strategy for the identification of d-Ins(1,4,5)P3 in higher plants. By the use of a short-term non-equilibrium labelling strategy in permeabilized plant protoplasts, coupled to the use of a 'metabolic trap' to prevent degradation of [32P]Ins(1,4,5)P3, we were able to determine the distribution of 32P in individual phosphate esters of Ins(1,4,5)P3. The [32]Ins(1,4,5)P3 identified showed the same distribution of label in individual phosphate esters as that of [32P]PtdIns(4,5)P2 isolated from the same tissue. We thus provide in vivo evidence for the action of a PtdIns(4,5)P2-directed PLC activity in plant cells which is responsible for the production of Ins(1,4,5)P3 observed here. This observation does not, however, exclude the possibility that in other cells or under different conditions Ins(1,4,5)P3 can be generated by alternative routes.

Distribution of mineral nutrient from nodal roots of Trifolium repens: Genotypic variation in intra‐plant allocation of 32P and 45Ca

To assess genotypic variability in nutrient supply of shoot branches, the distribution of 32P and 45Ca exported from a source nodal root (24‐h uptake period) was measured within a genotype of a large‐leaved (Kopu) and a small‐leaved (Tahora) cultivar of Trifolium repens. Source‐sink relationships of plants were modified by root severance, defoliation, and shade treatments. In control plants of both genotypes distribution of 32P and 45Ca closely followed the pathways that could be predicted from the known phyllotactic constraints on the vascular system. As such there was little allocation of radioisotopes (3.1% and 2.5% of exported 32P and 45Ca, respectively) from the source root to branches on the apposite side of the parent axis (far‐side branches). However, genotypic differences in nutrient allocation were apparent, when treatments were imposed to alter intra‐plant source‐sink relationships. In the large‐leaved genotype, the imposed treatments had minor effects on the allocation to far‐side branches: whereas, in the small‐leaved genotype, root severance and defoliation treatments increased lateral transport to far‐side branches to 30% (32P) and 10% (45Ca) of exported radioisotopes. Genotypes with low (8–9) and high (12–13) numbers of vascular bundles were selected from within the large‐leaved cultivar. Distribution of 32P was then measured after plants had been pre‐treated by removal of all far‐side roots two days prior to labelling. Genotypes with low vascular bundle number allocated 20% and those with high vascular bundle number 3.2% of exported 32P to far‐side branches. It was concluded (1) that genotypic variation exists within T. repens for potential to alter intra‐plant allocation of mineral nutrients, in response to treatments that modify source‐sink relationships within plants; and (2) that this variation is correlated with differences among genotypes in the organisation of the vasculature of their stolons.

Distribution of phosphorus and calcium from nodal roots of Trifolium repens: the relative importance of transport via xylem or phloem

summaryThe intra‐plant distribution of radioactive phosphorus (32P) and calcium (45Ca) exported from a single nodal root of Trifolium repens (white clover) was measured to ascertain if initial allocation of radioisotopes via the xylem was significantly altered over time by redistribution via the phloem. The radioisotopes were simultaneously supplied in a 2 h pulse which was followed by supply of non‐radioactive nutrient solution until harvest 3, 6, 9, 21 or 72 h after the start of labelling. By the end of the 3 d experimental period net export from the source root was 94 % total absorbed 45Ca but only 56 % of 32P. After movement through the stolon of the parent axis, main sinks for both nutrients were the leaf and the branch at the second node distal to the source root (64 and 30 % of total absorbed 45Ca and 32P respectively after 72 h). Very little of either radioisotope moved into branches located along the stolon of the parent axis, on the side opposite to the source root (1 and 2% of total absorbed 45Ca and 32P respectively after 72 h). This evidence, along with knowledge of the vascular architecture of the genotype, indicated that the allocation of mineral nutrients was determined chiefly by the vascular links between the source root and sink organs rather than sink size or proximity to the source root. Data indicated that some distribution of 32P occurred via the phloem but that it was quantitatively small in relation to allocation via the xylem both in the short (3 h) and medium (72 h) terms.

The self‐diffusion of strongly adsorbed anions in soil: a two‐path model to simulate restricted access to exchange sites

SummaryThe consequences of slow diffusion of strongly adsorbed solutes into soil aggregates are not fully understood. The distribution of 32P after diffusing down a soil column cannot be explained from a consideration of liquid‐phase impedance factor and isotopic exchange alone, as can that of 36CI.A model was developed that considers the soil to provide linked parallel intra‐aggregate and inter‐aggregate pathways. With this geometry, simulations agreed fairly well with experimental data when the intra‐aggregate impedance factor was 0.001, using other model parameters that had been determined in independent equilibration studies. With this intra‐aggregate impedance factor, the model yielded straight‐line plots for chloride and the overall impedance factor derived agreed closely with the experimental one. The intra‐aggregate impedance factor for 32P also agreed with the rate of reaction of the rapidly‐exchanging P fraction previously determined. The slowly‐exchanging P fraction has little influence on the P concentration profiles up to 10 d, but the small exchanged amount does have an effect at run times of 57 d.The slow intra‐aggregate diffusion of strongly adsorbed solutes decreases the amount adsorbed from a surface source of supply, and it also decreases the amount that is taken up by a surface sink. However, the interaggregate solution concentration remote from a source of supply is considerably increased, which may be important if the solute is a pollutant or a nutrient.

Phosphorylation of a second site for myosin light chain kinase on platelet myosin

The 20,000-dalton light chain of bovine platelet myosin is phosphorylated at two sites by myosin light chain kinase. The first and second phosphorylation sites are at a serine and a threonine residue, respectively. The location of the phosphorylation sites was determined by using limited proteolysis. The N-terminal sequence of the 17,000-dalton tryptic fragment of platelet myosin 20,000-dalton light chain was found to be identical with that of gizzard 20,000-dalton light chain from Ala-17 to Phe-33. On the basis of these results and the distribution of 32P among the proteolytic fragments, it was concluded that serine-19 and threonine-18 were the two phosphorylation sites. Phosphorylation at the threonine residue markedly increases the actin-activated ATPase activity of myosin. It was found that platelet myosin forms 10S and 6S conformations and its Mg2+-ATPase activity parallels the transition from the 6S to the 10S conformation. The conformational transition was influenced by phosphorylation at both sites, and the phosphorylation at the threonine residue further shifted the equilibrium toward the 6S conformation. The phosphorylation at the threonine residue also induced thick filament formation in the presence of ATP. These results suggest that the phosphorylation at the threonine residue as well as at the serine residue may play an important role in the contractility of nonmuscle cells.

Integrated pest control in forest management — Combined use of pheromones and insecticides for attracting and killing the Bark Beetle Ips typographus I. Studies with 32P-labelled methamidophos in the ascending sap of spruce

In 1984–1986, 32P-labelled methamidophos was used to investigate the translocation, distribution and breakdown of this systemic insecticide after penetration into the ascending sap of spruce ( Picea abies Karst.), following bark treatment with an adhesive paste of 15% active ingredient. The β-radiation of 32P permits a semi-quantitative measurement on the bark of standing trees as well as quantitative sample analysis and autoradiography. The following results are given: method of application and total 32P in the ascending sap; distribution of 32P in trunk cross-sections and in bark/needles; 32P in the killed beetles corresponding to the toxic dose, separation of methamidophos and breakdown products; halflife of breakdown in vitro and in vivo; and environmental implications. From the xylem, methamidophos penetrates into the phloem, but no 32P appears outside on the surface of the bark, so that only insects mining inside the bark are killed and no hazards for useful insects, birds and wildlife animals can arise. Traces of 32P were measured in air and water, and no 32P was found in the soil due to the translocation upwards only. The approximate oral toxicity of methamidophos to Ips typographus was estimated to be < 5 mg/kg and the half-life in spruce was 45 days (15°C), indicating a sufficient biological effectiveness over several months, preferably in areas of low average temperatures and in trees impaired by environmental pollution.

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long‐distance transport of phosphate. The effects of two hormones, gibberellic acid (GA3) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (32P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA3 treatment (10−4 M) of the tuber as well as of the leaves led to reduced transport of 32P into the tuber. By contrast, treatment of the tuber with ABA (10−4M) did not change the 32P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.

Effect of Phosphorus Deficiency on Source-Sink Interactions Between the Flag Leaf and Developing Grain in Barley

Chapin, F. S. Ill and Wardlaw, I. F. 1988. Effect of phosphorus deficiency on source-sink interactions between the flag leaf and developing grain in barley. —J. exp. Bot. 39: 165-177. Photosynthetic rate and stomatal conductance of P-deficient barley plants were increased by manipulations that increased plant demand for carbohydrates (shading of the ear or removal of other leaves). This was associated with retarded senescence of the flag leaf. Similarly, small flag leaves (i.e. those having large sinks relative to their own size) had high rates of photosynthesis. These relationships were most pronounced under conditions of P deficiency. Distribution of 32P and 14C from the flag leaf was determined by the carbohydrate demand of a plant part and was unaffected by altering the sink strength for P. Thus, ear shading increased accumulation by developing grains of 32P and 14C applied to the flag leaf, grain removal reduced accumulation by grain of both radioisotopes, and removal of lower leaves increased movement of both radioisotopes to vegetative plant parts; in contrast, P addition to the ear had no effect upon 32P or 14C distribution. P deficiency reduced the amount of tillering and, therefore, the movement of 32P and 14C to developing tillers and increased retention of 14C and 32P in the sheath of the flag leaf but otherwise had no major effect on patterns of carbon and phosphorus distribution. We conclude that the major effects of P deficiency upon source-sink interactions are (1) to reduce the number or activity of sinks in the shoot (grains and daughter tillers) and (2) to increase the sensitivity with which photosynthesis responds to demand for carbohydrate. The strengthened source-sink interaction under conditions of P deficiency provides a regulatory mechanism for reducing carbohydrate accumulation under conditions where carbohydrates do not strongly limit growth.

Insulin resistance in denervated skeletal muscle. Inability of insulin to stimulate dephosphorylation of glycogen synthase in denervated rat epitrochlearis muscles.

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.

Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.