Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.

Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid.

Free fatty acids in plasma and cells are mainly bound to membranes and proteins such as albumin and fatty acid binding proteins (FABP), which can regulate their biological activities and metabolic transformations. We have investigated the effect of FABP and albumin on the peroxidation of linoleic acid (18:2) and arachidonic acid (20:4) by 15-lipoxygenase (15-LO). Rabbit reticulocyte 15-LO produced a rapid conversion of [1-14C]18:2 to 13-hydroxyoctadecadienoic acid (13-HODE) and [3H]20:4 to 15-hydroxyeicosatetraenoic acid (15-HETE). 13-HODE formation was reduced when intestinal FABP (I-FABP). liver FABP (L-FABP) or albumin was added. The relative ability of these proteins to reduce 15-LO induced formation of 13-HODE and 15-HETE was BSA > L-FABP > I-FABP. Smaller reductions in activity were observed with 20:4 as compared to 18:2. The IC50-values of I-FABP and L-FABP, using either 18:2 (3.4 microM) or 20:4 (3.4 microM), were 4.6 +/- 0.6 and 1.9 +/- 0.2 microM, respectively, for reduction of 13-HODE and 6.8 +/- 0.3 and 3.1 +/- 0.2 microM, respectively, for reduction of 15-HETE formation. The smaller 15-HETE reduction correlated with decreased binding of 20:4 to the FABP. Titration calorimetry also showed that the I-FABP IC50 for 18:2, 0.25 microM, was lower then for 20:4, 0.6 microM. Thus the reduction in fatty acid lipid peroxidation relates to the binding capacity of each FABP. We also demonstrated that 18:2 rapidly diffuses (flip-flops) across the phospholipid bilayer of small unilamellar vesicles (SUV) and measured partitioning of 18:2 between proteins and SUV by the pyranin fluorescence method [Kamp, F. and Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Addition of proteins to SUV in buffer resulted in a complete desorption of 18:2 from SUV with a relative effect of BSA > L-FABP > I-FABP. This suggests that the relative effects of these proteins on 18:2 peroxidation will not be altered by the presence of membranes. Our results indicate that FAPBs protect intracellular polyunsaturated fatty acids against peroxidation and, through differential binding of 18:2 and 20:4, they may modulate the availability of these polyunsaturated fatty acids to intracellular oxidative pathways.

Molecular cloning and N-terminal analysis of bovine cystatin C. Identification of a full-length N-terminal region.

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Purification, characterization, and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.

Partial amino acid sequences of two proteins, purified from barley leaves reacting hypersensitively to the powdery mildew fungus, showed a high degree of amino acid identity to the PR-1 proteins originally described in tobacco. The proteins, subsequently designated HvPR-1a and HvPR-1b, show apparent pI values of approximately 10.5 and 11, respectively and apparent M(r) 15,000. Independently, differential screening of a cDNA library prepared from barley leaves, exhibiting a compatible interaction with the powdery mildew fungus, resulted in isolation of cDNA species representing two PR-1 homologs. With the exception of one amino acid, the partial amino acid sequences of HvPR-1a and HvPR-1b are identical to internal sequences of the polypeptides derived from the two cDNA species. These derived polypeptides are each 164 amino acids long and both have putative N-terminal leader sequences of 24 amino acids. That these proposed leader sequences are functional is indicated by the observed occurrence of both proteins in the intercellular fluid. The proposed mature proteins (calculated M(r) 14,490 and 15,204) share 91% identical amino acids with each other and 56 to 74% with other PR-1 proteins. Northern blot hybridization and immunoblotting, respectively, show that both transcripts and both proteins accumulate following inoculation of susceptible and hypersensitivity resistant barley leaves with the powdery mildew fungus.

Characterization of the 97 and 103 kDa forms of starch branching enzyme from potato tubers.

N-Terminal analysis, peptide mapping and partial peptide sequencing of the 97 and 103 kDa forms of starch branching enzyme from potato tubers showed that the two forms are highly related. A comparison with sequence data in the literature showed that these forms belong to the starch branching enzyme isoform I family. An internal cDNA fragment was obtained using PCR technology on potato tuber RNA with two oligonucleotide primers constructed from the peptide sequence data. Southern blot analysis using the PCR fragment as probe showed that there is only one gene locus encoding this isoform of the enzyme in Solanum tuberosum as well as in Solanum commersonii.

Reoxygenation-induced cell damage of isolated neonatal rat ventricular myocytes can be reduced by chain-breaking antioxidants.

To study the role of chain-breaking antioxidants on reperfusion injury in the ischemic heart, cultured ventricular heart cells (myocytes) were subjected to hypoxia and reoxygenation. The myocytes were prepared from neonatal rats and cultured in F10 medium that was supplemented with serum. As a marker for cell damage, lactate dehydrogenase was analyzed in the medium. Cells subjected to hypoxia for 5 h showed a 1.9 fold increase in lactate dehydrogenase (LD) leakage, while cells subjected to 1 h hypoxia followed by 4 h reoxygenation showed a 5-fold increase in LD intake. Alpha-tocopherol, beta-carotene, nordihydroguairetic acid (NDGA), butylated hydroxyltoluene (BHT), and ICI211965 were added to the cell medium every 24 h for 6 d prior to reoxygenation. All compounds protected against reoxygenation-induced cell damage. In the presence of the 5-lipoxygenase inhibitor ICI211965, protection against LD leakage was found only at high concentrations, which corresponded to the antioxidative effect of ICI211965, and not to inhibition of 5-lipoxygenase. We conclude that cultured ventricular myocytes can be used to evaluate the protective effect of antioxidants on reoxygenation-induced cell damage, and that chain-breaking antioxidants protected well against reoxygenation-induced cell damage.

Effects of a novel, low-molecular weight inhibitor of lipid peroxidation on ischemia-reperfusion injury in isolated rat hearts and in cultured cardiomyocytes.

We investigated the effect of H290/51, a novel, low-molecular-weight inhibitor of lipid peroxidation, on cardiac ischemia-reperfusion injury. Lactate dehydrogenase (LD) release from cultured cardiomyocytes exposed to 1 h hypoxia and 4 h reoxygenation was measured after pretreatment with different concentrations of H290/51. In another series, Langendorff-perfused rat hearts were exposed to 30 min global ischemia and 60 min reperfusion (n=minimum 10 in each group): 1. Control ischemia-reperfusion. 2. Vehicle throughout the experiment. 3. Vehicle during stabilization, and H290/51 (10(-6) mol/l) during reperfusion. 4. H290/51 throughout the experiments. During reoxygenation of isolated cardiomyocytes, H290/51 dose dependently inhibited LD release with an pIC50 value of 7.2+/-0.4 (mean+/-SEM), with 10(-6) mol/l as the lowest efficient concentration. In isolated hearts ischemia-reperfusion induced severe reperfusion arrhythmias, reduced left ventricular developed pressure (LVDP) and coronary flow (CF), and increased LV end-diastolic pressure (LVEDP). LD activity in the effluent increased. H290/51 throughout perfusion (group 4) reduced the occurrence of severe reperfusion arrhythmias (p < .0001), attenuated the decrease of LVDP (p < .008), and CF (p < .006), the increase of LVEDP (p < .008), and the release of LD (p < .002). Tissue contents of thiobarbituric acid-reactive substances did not increase during reperfusion in controls, but was reduced in group 4 (p < .004). H290/51 given only during reperfusion (group 3) tended to improve cardiac function, but significantly so only for increase of CF (p < .01). The lipid peroxidation inhibitor H290/51 attenuated cardiac injury induced by ischemia-reperfusion.

Muscarinic receptor coupling to inositol phospholipid metabolism in guinea-pig cerebral cortex, parotid gland and ileal smooth muscle.

Inositol phospholipid hydrolysis induced by agonist-stimulation of muscarinic receptors has been examined in slices of guinea-pig cerebral cortex, parotid gland and ileal smooth muscle. An assay measuring 3H-inositol phosphate formation from prelabelled lipids in the presence of LiCl, allowed marked stimulation by agonists to be followed. The pD2-value of carbachol differed markedly, between tissues being more than 10-fold lower in cerebral cortex than in parotid gland. The partial agonist oxotremorine showed the largest relative maximal responsiveness in parotid gland, followed by ileum and cortex. Atropine suppressed the phosphoinositide response to carbachol with an almost similar affinity in each tissue, but pirenzepine was found to have a 20-fold higher affinity in cerebral cortex, pKi = 7.7 than in parotid gland, pKi = 6.3. Carbachol, even in the presence of guanosine triphosphate (GTP), displayed complex binding against 3H-N-methylscopolamine (3H-NMS) in cortical and ileal membranes, though in membranes from the parotid gland a single homogeneous population was found. Atropine inhibition of 3H-NMS parallelled its suppression of the phosphoinositide response, the affinities in each tissue studied being similar. Pirenzepine inhibited binding from two components in cerebral cortex, the high affinity value being similar to that obtained in the phosphoinositide assay. In parotid gland, however, only low affinity pirenzepine binding sites were observed, closely resembling the affinity found for this antagonist in the functional assay. These experiments suggest (a) that there are differences between agonist occupation of muscarinic receptors and phosphoinositide hydrolysis within the different tissues, (b) that both high and low affinity pirenzepine binding sites appear to be linked to phosphoinositide metabolism, and (c) that low affinity pirenzepine sites may be more efficiently coupled to the hydrolysis of phosphoinositides.

Correlation between oxidation of low density lipoproteins and prostacyclin synthesis in cultured smooth muscle cells.

The effect of antioxidants on the oxidation of low density lipoproteins in relation to prostacyclin synthesis was investigated in the prescence of rabbit smooth muscle cells (SMC) and Fe-containing culture medium. The lipid peroxidation of low density lipoproteins (LDL) assayed as thiobarbituric acid reactive substances was increased from 0.5 to 1.4 nmol malondialdehyde/mL by the presence of smooth muscle cells. Two potent antioxidants, nordihydroguairetic acid (NDGA) and butylated hydroxytoluene (BHT), inhibited lipoprotein oxidation by IC50 values of 0.2 and 0.8 microM, respectively. Inhibition of lipoprotein oxidation was associated with an increased prostacyclin synthesis by the SMC, the effect being more pronounced with nordihydroguairetic acid than with butylated hydroxytoluene. The stable metabolite of the lipid hydroperoxide, 15-hydroxyeicosatetraenoic acid (15-HETE), formed in the 15-lipoxygenase pathway was measured following antioxidant treatment and found to be eliminated or greatly reduced by both antioxidants. The results presented show that lipid hydroperoxides, formed as a consequence of lipoprotein oxidation and promoted by the smooth muscle cells through a lipoxygenase reaction, may regulate prostacyclin synthase, a process which may be influenced by the addition of antioxidants.

The beta 1- and beta 2-adrenoceptor affinity of atenolol and metoprolol. A receptor-binding study performed with different radioligands in tissues from the rat, the guinea pig and man.

The radioligand binding technique was used to perform a systematic investigation of the beta 1- and beta 2-adrenoceptor affinity of atenolol and metoprolol in tissues from the rat, the guinea pig and man. Radioligands, [125I](+/-)hydroxybenzylpindolol, [125I](-)pindolol, [3H](-)dihydroalprenolol and [3H](-)CGP12177, with different degrees of lipophilicity were used in the binding experiments. In membrane preparations of rat ventricular myocardium and uterus, the number of specific binding sites was similar when comparing experiments performed with the different radioligands. The percentage of the beta 1- and beta 2-adrenoceptor subpopulations in the tissues studied was not dependent on the radioligand or displacing compound used. Furthermore, the affinity of metoprolol and atenolol for beta 1- and beta 2-adrenoceptors was independent of the radioligand used or the tissue studied. The beta 1-adrenoceptor affinity of metoprolol was about 6-7 times higher than that of atenolol, while the beta 1-adrenoceptor selectivity was similar (about 30-fold) for the two beta-blockers. It is concluded that the physical-chemical properties of the radioactive ligands and beta-blockers studied do not affect the results obtained from beta-adrenoceptor-binding experiments in cellular membrane fractions. The beta 1- and beta 2-adrenoceptor affinities did not change in any experiments performed in tissues from the rat, the guinea pig and man for either atenolol or metoprolol.

Measurement of superoxide anion production using maximal rate of cytochrome (III) C reduction in phorbol ester stimulated neutrophils, immobilised to microtiter plates.

In the present investigation, a method of studying the maximal rate of superoxide anion (O2.-) production in immobilised human neutrophils using a microtiter plate technique has been developed. The rate of O2.- production was determined from the rate of reduction of cytochrome (III) C, studied as the increase in absorbance at 550 nm. The protein kinase C activator, phorbol 12-myristate 13-acetate, was used to stimulate O2.- production. Neutrophils were evenly immobilised as a monolayer to microtiter culture plates to provide a reproducible exposure to the medium. Phorbol ester stimulated O2.- production was inhibited by staurosporine, a well-known inhibitor of protein kinase C, and by diphenylene iodonium, a potent NADPH-oxidase inhibitor, with IC50-values in this assay of 20 and 220 nm, respectively. The extracellularly produced O2.- was removed by superoxide dismutase with a half maximal effect of 0.6 microgram/mL. The maximal production rate of O2.- could therefore be estimated by addition of 20 micrograms/mL superoxide dismutase. Several antioxidants, including butylated hydroxytoluene, nordihydroguairetic acid, probucol and alpha-tocopherol, were studied and showed neither an effect on O2.- production nor a scavenging effect. This new method was highly reproducible, and the continuous measurement of O2.- production was very useful for validating the effect of inhibitors. The developed microtiter technique using immobilised cells has a large capacity and allows different compounds to be tested under comparable conditions, since they are exposed to the cells in a similar way. This is also the first test model which describes O2.- production as the maximal rate of cytochrome (III) C reduction.

The quantity of protein-bound [32P]phosphotyrosine in hepatocytes and fibroblasts. The effects of tyrosine protein kinase activating agents.

Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.

Phosphorylation of CaBP1 and CaBP2 by protein kinase CK2.

Several proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P]ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were grown in the presence of [32P] phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 in microsomes.

Analysis of proteins in the extracellular matrix of the plant pathogenic fungus Bipolaris sorokiniana using 2-D gel electrophoresis and MS/MS.

A method was developed for isolating and sequencing proteins present in the extracellular matrix (ECM) of germlings and hyphae of filamentous fungi. Surface proteins of the cereal pathogen Bipolaris sorokiniana were labelled with a membrane impermeable biotinylating agent and extracted using a glycine-HCl buffer. Extracted proteins were purified by affinity binding to streptavidin-conjugated magnetic beads or by two-dimensional gel electrophoresis. Four of the biotinylated proteins from the ECM of B. sorokiniana were isolated, in gel digested with trypsin and partly sequenced by tandem mass spectrometry. No significant sequence similarities to proteins in databases were obtained.

Characterization of the beta-adrenergic inhibition of motility in cat colon strips.

The inhibition of spontaneous contractile activity in isolated cat colon strips by isoprenaline, prenalterol and terbutaline was studied. Isoprenaline produced a concentration-dependent complete inhibition with an EC50 of 6.18 nM. Prenalterol and terbutaline produced a concentration-dependent partial inhibition of 68 and 75% respectively, with EC50 of 0.52 and 0.43 microM respectively. The inhibitory effects of the three agonists were blocked by metoprolol and IPS 339 in such a way as to suggest that the prenalterol effect was mediated by beta 1-adrenoceptors and the terbutaline effect was mediated by beta 2-adrenoceptors, whereas the effect of isoprenaline appeared to be mediated by both types of receptors. Thus, the results indicate that both beta 1- and beta 2-adrenoceptors are involved in the adrenergic inhibition of spontaneous contractile activity in the cat colon strip. Qualitative effects of prenalterol and terbutaline on the spontaneous contractile activity in the cat colon strip indicated that the beta 1-adrenoceptor-mediated effects may be related to inhibition of the activity in cholinergic neurons, whereas the beta 2-adrenoceptors may be acting on the smooth muscle cells per se.

The 1,4-beta-D-glucan glucanohydrolases from Phanerochaete chrysosporium. Re-assessment of their significance in cellulose degradation mechanisms.

A physico-chemical, functional and structural characterization, including partial sequence data, of three major 1,4-beta-D-glucan glucanohydrolases (EC. 3.2.1.4) isolated from the culture filtrate of the white-rot fungus Phanerochaete chrysosporium, shows that all three enzymes belong to a single family of cellulases. EG44, pI 4.3, (named after its apparent molecular mass in kDa), shows a clear homology with Schizopyllum commune Endoglucanase I (EGI); whereas EG38, pI 4.9, (named in the same manner) is related more closely to Trichoderma reesei (Trichoderma longibrachiatum) Endoglucanase III (EGIII). EG36, pI 5.6-5.7, is probably an EG38 protein lacking its cellulose binding domain. Strong synergistic action is induced by the enzymes acting in concert with cellobiohydrolases (CBHI and CBHII) from the same organism, indicating a highly effective enzymatic system for cellulose degradation. Controlled proteolysis with papain has allowed a so far unique cleavage of endoglucanases EG44 and EG38 into two domains: a core protein, which virtually lacks the capacity to absorb onto microcrystal-line cellulose but retains full catalytic activity against carboxymethyl cellulose and low molecular weight soluble substrates; and a peptide fragment corresponding to the cellulose binding domain. The latter appears to be of paramount significance in the mechanisms involved in the hydrolysis of microcrystalline cellulose.

Phosphorylation of sucrose synthase from maize seedlings.

Two isoforms of sucrose synthase (SS1 and SS2) from maize (Zea mays, var. Mona) seedlings co-purified with a calcium and phospholipid dependent protein kinase. The enzymatic preparation obtained gave a positive reaction with the antibody against mammalian protein kinase C. Maize sucrose synthase was phosphorylated by the endogenous protein kinase. Also, mammalian protein kinases (protein kinase C and protein kinase A) were able to phosphorylate the 86 kDa subunit of sucrose synthase. When excised seedlings were fed [32P]orthophosphate, sucrose synthase was also phosphorylated. Microsequencing of in vivo labelled enzyme has shown phosphorylation of Ser-15 in SS2. The present work provides evidence that maize sucrose synthase is the physiological substrate of the endogenous calcium and phospholipid dependent protein kinase(s).

Muscarinic receptor interaction with full and partial beta-adrenoceptor agonists in the rat colon strip.

beta-Adrenoceptor agonists inhibit contractile activity in isolated colon strips. In order to demonstrate that beta-adrenoceptors are located at different functional levels within the colon wall, increasing concentrations of muscarinic agonists were used to interact functionally with the beta-adrenoceptor-mediated inhibition of spontaneous colon activity. The effects of the full agonists isoprenaline and terbutaline and of the partial agonist prenalterol were functionally antagonized by carbachol (0.03 and 0.3 mumol/l) and bethanechol (1.3 and 30 mumol/l). This functional antagonism was parallelled by an increase in baseline tension and spontaneous contractile activity of the isolated colon strip. At lower concentrations of carbachol (0.003 and 0.01 mumol/l) or bethanechol (0.03 and 0.3 mumol/l) no effect on the contractile status of the smooth muscle or on the pD2-values of the full agonists was seen. However, at these lower concentrations of muscarinic agonists a marked decrease in the maximal inhibitory response to the partial beta-adrenoceptor agonist prenalterol was demonstrated. The inhibitory response to prenalterol showed a biphasic concentration-response curve. The muscarinic antagonist atropine produced an increase in the maximal response of the high potency component of the concentration-response curve for prenalterol and an increase in the sensitivity to isoprenaline. These results demonstrate the presence of a high cholinergic tone in the colon preparation of a magnitude that clearly reduces the sensitivity to beta-adrenoceptor agonists. The different responses to full and partial beta-adrenoceptor agonists in the presence of increasing concentrations of muscarinic agonists may indicate that beta-adrenoceptors are located on two different functional units within the colon wall.