Particulate Preparations Research Articles

FACTORS INFLUENCING PERTECHNETATE UPTAKE BY RABBIT KNEES

The accumulation of radioactivity over rabbit knees immediately after the injection of 99mTc-pertechnetate or one of two 99mTc-labelled particulate preparations has been measured in animals with and without experimental arthritis. The results indicate that in inflamed, but not normal joints, a peak of radioactivity is detectable within 9 seconds of injection and suggest that the rate of accumulation of radioactivity over the joint may be a more sensitive indicator of inflammatory activity than measurement at a fixed time. Clearance from the circulation and tissue distribution of the three radioactive preparations were also measured.

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (B max) of 40 ± 8 fmol/mg protein with an apparent dissociation constant (K d) of 6.0 ± 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The K d value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The B max of 60 ± 10 fmol/mg protein and the K d of 5 ± 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 μM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, tyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101–126) binding sites was rANF (92–126) > rANF (101– 126) > rANF (99–126) > rANF (103–126) > Tyr o − Atriopeptin I > hANF (105–126) > rANF (1–126) . Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requeriments. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demostration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.

Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: Effects of neurotransmitters and depolarization

The specific binding of [3H] and [32P]Ins(1,4,5)P3 to a particulate preparation of bovine adrenal cortex has been used as a radioreceptor assay to determine the concentration of Ins(1,4,5)P3 in agonist- and depolarization-stimulated rat cerebral cortex slices. The resting concentration of Ins(1,4,5)P3 in slices that had been preincubated in a physiological medium was 18.8 +/- 2.6 pmol/mg prot. Carbachol evoked a rapid and dose-related increase in the concentration of Ins(1,4,5)P3. Maximal stimulation (80%) was already seen at the earliest point (10 sec) examined and was maintained for at least 5 min. The EC50 for carbachol was 75 +/- 17 microM and the response was totally suppressed by the muscarinic antagonist atropine. A direct comparison in the same slices was made between mass determinations and [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 accumulation determined by h.p.l.c. Although an identical time course was observed for cold and radiolabelled Ins(1,4,5)P3, the greater stimulation of [3H]Ins(1,4,5)P3 may indicate changes in specific radioactivity. Of a variety of other receptor agonists studied, only the glutamate receptor agonist quisqualate, and noradrenaline significantly increased the mass of Ins(1,4,5)P3 in cerebral cortical slices. However, depolarizing concentrations of K+ were as effective as carbachol at elevating this second messenger.

Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

We describe unique, high-affinity binding sites for omega[125I]conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

Human platelet phospholipase A 2 activity is responsive in vitro to pH and Ca 2+ variations which parallel those occurring after platelet activation in vivo

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A 2 enzyme species whose activity is regulated by Na +/H + exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660–8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197–9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na +/H + antiporter enables the phospholipase A 2 enzyme to function at ambient or low concentrations of intraplatelet Ca 2+. To test the hypothesis that the Ca 2+ dependence of platelet phospholipase A 2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca 2+ dependence of this enzyme as a function of changes in pH (from pH 6.8–8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A 2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca 2+ (EC 50 1–2 μM) and plateaued above 10 μM Ca 2+. Enzymatic activity measured at 4.8 μM Ca 2+ was increased by raising the pH from 5.5 to 8.0 (EC 50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca 2+ concentrations. The interdependent regulation of phospholipase A 2 activity by changes in pH and Ca 2+ suggests that phospholipase A 2 could serve to integrate changes in intracellular pH and available Ca 2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.

Rapid Deactivation of NADPH Oxidase in Neutrophils: Continuous Replacement by Newly Activated Enzyme Sustains the Respiratory Burst

The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N-ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.

Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst

The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N- ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N- ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N- ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.

Iron-sulfur centers involved in methanogenic electron transfer in methanobacterium thermoautothrophicum (delta-H)

Utilizing a subcellular particulate preparation from Methanobacterium thermoautotrophicum (delta-H) which contains all detectable methanogenic electron transfer activity, we present the results of the effects of the anaerobic addition of oxidized factor F420 and of methyl coenzyme M plus ATP on the EPR signals from reduced iron-sulfur centers and a rapidly-relaxing radical species. Based on these results, we report the existence of a minimum of three iron-sulfur centers which are capable of donating electrons to these cofactors.

125I-Luteinizing Hormone (LH) binding to soluble receptors from the primate ( [formula omitted]) corpus luteum: Effects of ethanol exposure

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30m min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p<0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p<0.05). Conditions which enhanced LH binding to particulates, i.e., 1–8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.

Diacylglycerol acyltransferase in maturing safflower seeds: its influences on the fatty acid composition of triacylglycerol and on the rate of triacylglycerol synthesis

Diacylglycerol acyltransferase in a particulate preparation from maturing safflower seeds showed no strict selectivity for acyl-CoA when acyl-CoA substrates were administered as mixtures. This suggested that the fatty acid composition of position 3 in safflower triacyl- sn-glycerol exclusively depends on the acyl-CoA composition in the cell. The specific activity of the acylation was approximately 3 nmol/min per mg protein of the preparation under the optimum assay conditions. This low activity and other data appeared to indicate that the diacylglycerol acyltransferase reaction may be the rate-limiting step in triacylglycerol synthesis in vivo.

Solubilization of the NADPH-oxidase from bone marrow derived macrophages by dialkylphospholipid and purification by HPLC-anion exchange chromatography.

The microsomal NADPH-oxidase of bone marrow-derived macrophages was partially purified by HPLC-anion exchange chromatography. Particulate preparations were solubilized by the Dialkylphospholipid 2-O-methyl-1-O-octadecyl-rac-glycero-3-phosphocholine. The enzyme was enriched up to 61-fold and proved to be stable under the assay conditions. The NADPH-oxidase depended on NADPH as electron donor with a Km of 2.1 microM, whereas no O2- -production was detected with up to 40 microM NADH. Ca2+ was found to be a strong inhibitor of the enzyme, whereas Mg2+ had no influence. O2- was determined by the very sensitive and selective method of superoxide dismutase-inhibitable chemiluminescence of lucigenin.

The oxidative decarboxylation of ornithine by extracts of higher plants

Extracts of wheat leaves catalysed the oxidative decarboxylation of ornithine in the presence of pyridoxal phosphate, with the production of 4-aminobutanamide and 4-aminobutyric acid. In similar experiments, putrescine was not converted to the amide or acid suggesting it is not an intermediate. The optimum concentration of pyridoxal phosphate was 1 mM, and activity declined on increasing the concentration to 10 mM. The K m for ornithine was 10 mM. The activity was found principally in the particulate fraction. Both the particulate and the soluble fractions showed two pH optima, at ca pH 9 and 10.5. Two pH optima were also found for ornithine decarboxylation in the particulate fraction of oat leaves: one at pH 6.5-7 and another at pH 9–9.5. Activity was lost on heating for 1 hr at 100°, though discontinuities in the thermal denaturation curve indicated a heterogeneous system. d- and l-Ornithine and l- lysine acted as substrates. 2,3-Diaminopropionic acid and 2,4-diaminobutyric acid were oxidized to ninhydrin-positive compounds which were presumably respectively 2-aminoacetamide and 3-aminopropanamide. Aminoguanidine (0.25 mM inhibited ornithine decarboxylation by 30–40%, while difluoromethylornithine at 1 mM showed no inhibition. Dithiothreitol, dithioerythritol, cysteine, mercaptoethanol and glutathione inhibited the oxidation at 1 and 5 mM. Particulate preparations of barley, sugar beet and rape leaves also oxidized ornithine to 4-aminobutanamide and 4-aminobutyric acid. Gel filtration on Sephadex G-25 showed that the M r of the major component causing oxidative decarboxylation of ornithine present in the wheat leaf supernatant was ca 4000. However another less active fraction was found to have a M r of > 5000. Mn 2+ ions at μM concentrations catalyse a similar decarboxylation of ornithine in the presence of pyridoxal phosphate with the production of 4-aminobutanamide, and may be responsible for the activity in the plant extracts. Mn 2+ ions are known to associate with nucleic acids and may therefore simulate a complex catalyst of relatively large M r . At high pH, Mn 2+ ions exist as the unstable hydroxide which can be readily oxidized, explaining the apparent thermal denaturation, which appears to be accelerated in the presence of the plant extracts.

A biochemical chimera suggesting the existence of at least two dolichol-P-glucose:Dolichol-P-P-oligosaccharide glucosyltransferases

As previously reported, incubation of liver dolichol-P, UDP-[ 14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[ 14C]Gal ( P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205–212 ). It is now reported that upon incubation of the latter with rat liver microsomes, [ 14C- galactose]-Gal 1Man 9GlcNAc 2-P-P-dolichol and [ 14C- galactose]Gal 1Glc 1Man 9GlcNAc 2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal 1Glc 2Man 9GlcNAc 2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix.

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.

1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol.

A 20,000 X g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1-acyl-sn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-1 mg protein-1. Although this preparation was also capable of catalyzing the acylation of sn-glycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acyl-CoA. The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl greater than palmitoleoyl greater than elaidoyl greater than cis-vaccenoyl greater than stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1-acyl-glycerol-3-P molecular species in the following order of effectiveness: linoleoyl = oleoyl greater than palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1-acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1-acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.

Stereospecific recognition sites for [ 3H]inositol(1,4,5)-trisphosphate in particulate preparations of rat cerebellum

A very high density of stereospecific binding sites for inositol(1,4,5)P 3 have been identified in rat cerebellar membranes using [ 3H]inositol(1,4,5)P 3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (K D 23 nM). Incubations were carried out at 4° and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [ 3H]-(1,4,5)P 3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P 3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P 3 was the only other phosphate to show relatively high affinity (K D 1500 nM). HPLC-pure Ins(1,3,4)P 3 and Ins(1,3,4,5)P 4 were substantially weaker and Ins(1,4)P 2, Ins-2-P 1, Ins-1-P 1, Ins(1,2)-cyclic P 1 and inositol were totally inactive at concentrations <50 μM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P 3 can initiate the release of bound Ca 2+.

Effect of N6-(L-2-phenylisopropyl)adenosine and insulin on cAMP metabolism in 3T3-L1 adipocytes.

The antilipolytic effect of N6-(L-2-phenylisopropyl)-adenosine (PIA), an adenosine analogue thought to act via cell surface receptors, was investigated in 3T3-L1 adipocytes. PIA (1 microM) was as effective as 1 nM insulin in reducing lipolysis stimulated by 1 nM isoproterenol and more effective than insulin at higher isoproterenol concentrations. In intact adipocytes, PIA reduced isoproterenol-induced cyclic AMP (cAMP) accumulation and increased particulate cAMP phosphodiesterase. In particulate preparations PIA suppressed isoproterenol stimulation of adenylate cyclase. PIA was more effective than 5'-N-ethylcarboxamide adenosine (NECA) or adenosine in inhibiting adenylate cyclase and activating phosphodiesterase. In intact adipocytes, two agents with so-called "insulin-like" activities, i.e., anti-insulin receptor antibodies and wheat germ agglutinin (WGA), also increased particulate cAMP phosphodiesterase. Pertussis toxin, which inhibits stimulation of the particulate cAMP phosphodiesterase by insulin (but not by isoproterenol), also inhibited the effects of PIA, anti-insulin receptor antibodies, and WGA. In 3T3-L1 cells, PIA appears to inhibit lipolysis by inhibiting adenylate cyclase and stimulating phosphodiesterase; these effects of PIA, as well as those of anti-insulin receptor antibodies and WGA on phosphodiesterase, may be mediated via guanyl nucleotide-binding proteins.

TRANSPORT OF SUGAR IN PHYTOPHTHORA PALMIVORA (BUTL.) BUTL.

SummaryThe kinetics of uptake of sucrose, glucose and fructose by Phytophthora palmivora are all biphasic. At low concentrations, glucose and fructose share a common, high‐affinity, hexose carrier, and sucrose is probably hydrolyzed extracellularly at these concentrations before the transport of the resultant hexoses by this carrier. At higher concentrations, a low‐affinity carrier may transport sucrose directly, but this carrier appears to have a higher affinity for glucose than sucrose. Activity of neutral invertase (β‐D‐fructofuranoside fructohydrolase) in both soluble and particulate (cell‐wall) preparations is considerably in excess of the highest rates of sucrose uptake.

Protein phosphorylation and tyrosine kinase activity in medullary thyroid carcinomas of the rat.

The kinetics of endogenous protein phosphorylation and resultant phosphoprotein patterns were investigated in well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. Cytosolic or particulate fractions from these tumors were incubated with gamma-32P-ATP in the presence of various effectors. Phosphorylation appeared to be predominantly independent of exogenously added cyclic AMP. Magnesium and manganese were equally effective cofactors. For both tumor types 32P incorporation into cytosolic proteins was maximal within 3-4 min after addition of ATP and subsequently decreased gradually within 1 h. In contrast, in particulate preparations maximal incorporation was reached within 30 s and remained constant over a relatively long time span. In both cases, however, 32P incorporation in extracts from DMTCs were 50-100% higher as compared to AMTCs. Comparison of the phosphoprotein patterns of each tumor after in vitro phosphorylation showed some significant differences. A phosphoprotein with molecular weight of 90 kilodalton (90 kD) was exclusively expressed in the cytosol of DMTC, whereas 99- and 84-kD phosphoproteins were only present in the cytosol of AMTC. The DMTC particulate fraction contained two phosphoproteins (with molecular weights of 40 and 37 kD), which were absent from that of AMTC. In addition, a number of proteins were more intensely phosphorylated in one of the tumors, e.g. proteins of 94 and 33 kD in DMTC cytosol and a protein of 78 kD in AMTC cytosol. Calcium induced phosphorylation of five proteins in DMTC cytosol (with molecular weights of 69, 55, 49, 43 and 32 kD), which were less intensely or not phosphorylated in AMTC. Tyrosine kinase activity was investigated using exogenously added poly(glutamine:tyrosine, 4:1) as an artificial substrate. Cytosolic tyrosine kinase activity in DMTC was +/- 50% higher than in AMTC (11.9 +/- 0.6 and 8.2 +/- 1.7 pmol/mg/min, respectively). The enzyme activities in the particulate preparations were much higher than in the cytosols (+/- 100 pmol/mg/min), although considerable variations in enzyme activity between different tumors of either type were observed. Quantitative differences in tyrosine kinase activity between AMTC and DMTC particulate fractions did not seem to exist using this substrate. Phosphoamino acid analysis of endogenously phosphorylated proteins in both AMTC and DMTC showed phosphotyrosine to be present only in cytosolic proteins within the 50-kD molecular weight region, the majority of 32P being on serine and some on threonine.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucomannan synthase from suspension cultures of Pinus sylvestris

The activity of a glucomannan synthase was measured in particulate preparations obtained from suspension cultured cells of Pinus sylvestris. The product was characterized and found to correspond to the β 1 → 4 linked glucomannan present in the pine secondary wall. The specific activity of the synthase was found to increase in culture conditions favouring tracheid formation.