Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

Membrane perturbation by mastoparan 7 elicits a broad alteration in lipid composition of L1210 cells

Mastoparan 7 (Mas-7), an amphiphilic peptide possessing membrane perturbing activity, has been known to selectively stimulate some lipases. To examine changes in the lipid composition induced by Mas-7, we carried out systemic lipid analysis of L1210 cells after Mas-7 treatment. The total lipid was determined by HPLC, gas-liquid chromatography, and electrospray ionization mass spectrometry in conjunction with differential radiolabelling with [(32)P]orthophosphate, [(3)H]myristic acid, and [(3)H]arachidonic acid. The lipid analysis revealed multiple changes in more than 10 lipid classes. Free fatty acids (FFAs) and phosphatidylethanol (PEt), the phospholipase D product in the presence of ethanol, were increased significantly and phosphatidylcholine (PC) was decreased. Digitonin, a membrane permeabilizing reagent, similarly affected the lipid composition of L1210. The FFA released showed a very broad distribution of saturated, monounsaturated, and polyunsaturated fatty acids, implying that phospholipase A(2) alone could not account for all of the FFAs released. By comparing the molecular species of PEt with those of endogenous PC, we showed that phospholipase D in L1210 cells appeared to act selectively on diacyl-PC. The perturbation-induced alterations in the lipid composition brought about by Mas-7 might play a crucial role in the physiology of the affected cells.     

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

The turnover of myelin phospholipids in the adult and developing rat brain

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.     

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.     

Inorganic polyphosphate as an integral part of alkaline phosphatase preparations.

Alkaline phosphatase (from chicken intestinal sources) was shown to contain a considerable amount of polyanionic phosphorus which was released by basic digestion. The polyanionic phosphorus of alkaline phosphatase is not associated with protein or polyalcohols and does not exhibit a visible or ultraviolet absorption spectrum. Alkaline phosphatase and abiogenic inorganic polyphosphate were found to incorporate 32P-orthophosphate under similar experimental conditions. It has been previously reported that this enzyme will incorporate 32P-orthophosphate into its protein phosphoserine without the apparent concomitant utilization of an energy source. This reported phosphorylation was immediately reversible upon dilution of the phosphorylated enzyme with unlabelled orthophosphate, which indicates that the initial phosphorylation was an exchange reaction. These observations suggest that this polyanionic phosphorus from alkaline phosphatase may be inorganic polyphosphate.     

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.     

Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils

The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.     

Metabolism of phospholipids and the characterization of fatty acids in bovine corpus luteum

1. Phosphatidylcholine was the predominant phospholipid in bovine corpora lutea; it accounted for about 50% of the total phospholipid phosphorus. Phosphatidylethanolamine (13%) and ethanolamine plasmalogen (8-9%) were the next two major components. 2. After incubation of the tissue with [(32)P]orthophosphate the total radioactivity and specific radioactivity of phosphatidylinositol were higher than those of any other lipid. 3. Luteinizing hormone failed to increase significantly the incorporation of [(32)P]orthophosphate into total phospholipids from luteal tissue slices, but did stimulate progesterone synthesis and lactate production. 4. The proportion of oleate (18:1) in the neutral lipids and phospholipids was higher than that of any other fatty acid. 5. The proportion of unsaturated fatty acid in the tissue lipids exceeded 60%, and almost half of this was polyunsaturated. Arachidonate (20:4), docosatetraenoate (22:4) and docosapentaenoate (22:5) were the principal polyunsaturated fatty acids. 6. After incubation of luteal tissue with [1-(14)C]acetate, the greatest proportion of radioactivity in the fatty acids isolated from the total lipid fraction was in palmitate (16:0) and docosatetraenoate (22:4). Polyunsaturated fatty acids accounted for almost 50% of the (14)C radioactivity incorporated and this pattern was observed in phospholipids, triglycerides and free fatty acids.     

Lysosomal phospholipases A1 and A2 of bovine adrenal medulla

1. [(32)P]Lecithin and [(32)P]phosphatidylethanolamine were prepared by incubating rat liver mince with [(32)P]phosphate. With these (32)P-labelled phospholipids conditions for the quantitative assay of phospholipase A activity were established. 2. The distribution of phospholipase A activity between subcellular fractions of the bovine adrenal medulla was determined. Phospholipases A(1) and A(2), with pH optima at 4.2 and 6.5 respectively, were found in the large-granule fraction. By means of sucrose-density-gradient centrifugation it was shown that both these phospholipases were localized in lysosomes. 3. Lysosomal phospholipase A(1) catalysed the hydrolysis of [(32)P]lecithin and [(32)P]phosphatidylethanolamine at the same rate. The enzymic activity was inhibited by 70% in the presence of 10mm-calcium chloride. 4. Lysosomal phospholipase A(2) catalysed the hydrolysis of [(32)P]phosphatidylethanolamine more rapidly than it hydrolysed [(32)P]lecithin. The hydrolysis of [(32)P]phosphatidylethanolamine, but not that of [(32)P]lecithin, by phospholipase A(2) was activated by 0.8mm-calcium chloride. However, the hydrolysis of both substrates was inhibited by 8mm-calcium chloride. 5. The significance of the presence of phospholipase activity in lysosomes is discussed in relation to the functions of lysosomes in general and in the adrenal medulla.     

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.     

Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

Concerning the chemical nature of tubulin subunits that cap and stabilize microtubules

There is no definitive evidence on the nature of the cap at microtubule ends that is responsible for dynamic instability behavior. It was, therefore, of interest that steady-state microtubules assembled in 20 mM P(i) buffer and pulsed for 15-60 min with [gamma-(32)P]GTP contained approximately 26 [(32)P]P(i)/microtubule [Panda et al. (2002) Biochemistry 41, 1609-1617]. It was concluded that microtubules are capped with a tubulin-GDP-P(i) subunit at the end of each its 13 protofilaments and that this is responsible for stabilizing microtubules in the growth phase. Also, because microtubules with [(32)P]P(i) were isolated despite the presence of 20 mM P(i), it was concluded that P(i) in terminal tubulin-GDP-P(i) subunits does not exchange with solvent. These observations are inconsistent with our finding that tubulin-GDP-P(i) subunits do not stabilize microtubules and with evidence that the nucleotide, and presumably also P(i), in subunits at microtubule ends exchanges with solvent. We have resolved this discrepancy by finding that during the pulse period the added [(32)P]GTP was almost quantitatively hydrolyzed. The so-formed [(32)P]P(i) labeled the 20 mM P(i) buffer, and this exchanged into tubulin-GDP subunits in the core of the microtubule. Evidence for this was our finding of virtually identical [(32)P]P(i) in microtubules pulsed with [(32)P]GTP with a specific activity that varied 11-fold by using either 100 or 1,100 microM GTP in the reaction. Label uptake was insensitive to the [(32)P]GTP specific activity because in both cases hydrolysis generated 20 mM [(32)P]P(i) with a virtually identical specific activity. Also, approximately 0.4 mol of [(32)P]P(i) /tubulin dimer was found in microtubules when steady-state microtubules in 20 mM P(i) were pulsed with a trace amount of [(32)P]P(i). This stoichiometry is consistent with a 25 mM K(d) previously reported for P(i) binding to tubulin-GDP subunits in microtubules. It is concluded that, under the conditions used for the [(32)P]GTP pulse labeling, (32)P was incorporated into the entire microtubule from [(32)P]P(i) released into the solution, rather than into a tubulin-GDP-P(i) cap, from [(32)P]GTP. Thus, there is no evidence that tubulin-GDP-P(i) subunits accumulate in and stabilize microtubule ends.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.     

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.     

Studies on 5alpha-androst-16-en-3-one binding to porcine serum, plasma and testicular cytosolic fraction and to human serum

The present study evaluated whether a specific androstenone-binding protein is present in porcine and human serum, and in the cytosolic fraction of porcine testis. The binding of [(3)H]-androstenone to serum and testicular cytosol was measured in the absence (total binding) and presence (non-specific binding) of unlabelled androstenone. The optimization of the assay is described. As a part of the assay validation, the binding of [(3)H]-dihydrotestosterone ([(3)H]-DHT) to porcine and human serum was also examined. As expected, specific binding of [(3)H]-DHT was detected in human serum, but not in porcine serum. No specific androstenone-binding protein was detected, either in porcine or human serum, or in the cytosolic fraction of porcine testis. The amount of non-specific binding of [(3)H]-androstenone was slightly lower in porcine serum compared to human serum. Between-animal variations in [(3)H]-androstenone binding were studied in plasma samples from 15 animals with androstenone concentrations ranging from 1.1 to 23.1 ng/mL. Mean values+/-standard deviations of binding in these samples were 15.2+/-0.9% for total binding and 15.9+/-0.8% for non-specific bindings. Low between-animal variations indicate that androstenone binding does not affect androstenone accumulation in fat.     

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [gamma-(32)P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30-100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate (K(m)=1.6-5.6 microM) and capability to phosphorylate minor lipids using [(32)P]orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60-80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Determination of basal and stimulated levels of inositol triphosphate in [32P]orthophosphate-labeled platelets

Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1-4 and 10-30 pmol/10(8) cells, respectively.