Opioid receptor labeling with the chloromethyl ketone derivative of (3)H-Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) I: Binding properties of 3H-Tyr-D-Ala-Gly-(Me)Phe chloromethyl.

We prepared a tritiated chloromethyl ketone derivative of Tyr-D-Ala-Gly(Me)Phe-Gly-ol 3H-D-Ala-Gly-(Me)Phe-chloromethyl ketone, and studied its binding characteristics in rat brain membranes. A significant portion (about 70%) of the binding becomes wash-resistant after 60 min of incubation. The binding of the ligand is highly stereospecific and mu-opioid receptor selective. These characteristics of the ligand, together with its high specific radioactivity (57 Ci/mmol) makes it a good candidate for biochemical characterization and covalent labeling of mu opioid receptors.     

N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.     

3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.     

Effect of N,alpha-tosyl-L-phenylalanine chloromethyl ketone on measles virus P and M polypeptides

Treatment of measles virus-infected cells with 1 mM N,alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) was observed to change the polyacrylamide gel migration of viral polypeptides P and M. Untreated cells contained P as a mixture of P1 (70,000 daltons) and P2 (65,000 daltons) and M as a 38,000-dalton band M1 and a slightly smaller band, M2. TPCK treatment resulted in conversion of P largely to the 65,000-dalton band and of M to M1 and a slightly slower-migrating band. This effect could also be demonstrated by treating homogenates of infected cells with TPCK, and the evidence suggests that the compound reacts directly with the viral P and M polypeptides and thereby changes their gel migration. TPCK also inhibited measles virus-associated protein kinase, and treatment of virion preparations with the compound resulted in a loss of infectivity; however, it was not possible to directly correlate the inhibitory effect on these two biological functions with the change seen in polypeptides P and M.     

N-α-Tosyl-L-lysine chloromethyl ketone and N-α-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-α-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca²⁺ and phospholipid. TLCK (IC₅₀= 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-α-tosyl-L-phenylalanine chloromethyl ketone) (IC₅₀=8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.Protein kinase CTumor promotionProtease inhibitor     

Characterization of glucocorticoid receptors in S49 mouse lymphoma cells by affinity labeling with [3H]dexamethasone 21-mesylate

Glucocorticoid receptors in wild type and mutant S49 mouse lymphoma cells were affinity labeled with [3H]dexamethasone 21-mesylate and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of receptors in cytosol from wild type cells and nuclear transfer decreased (nt-) mutants was 97,000 (97 kDa). The molecular weight of receptors in cytosol from nuclear transfer increased (nti) mutants was 48 kDa. The 97 kDa receptor in cytosol from wild type cells was digested by chymotrypsin to a 40 kDa steroid-binding receptor fragment but the 48 kDa receptor in cytosol from nti mutants was resistant to digestion by chymotrypsin. In addition to the 48 kDa receptor, cytosol from nti mutants contained 40 and 18 kDa receptor fragments. Cytosol from the nt- mutants also contained 18 kDa receptor fragments. The 40 and 18 kDa receptor fragments were present in multiple subclones of a nti mutant cell line. Formation of these receptor fragments was not prevented by protease inhibitors and was not increased by extended incubation of cytosol samples. Both 48 and 40 kDa forms of the receptor, but not the 18 kDa form, could be activated and bound by DNA-cellulose.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.     

Metabolic labeling of Dirofilaria immitis third- and fourth-stage larvae and their excretory-secretory products.

Infective third-stage larvae of Dirofilaria immitis were collected from Aedes aegypti and cultured in vitro to the fourth stage. Larval proteins were labeled metabolically using [35S]cysteine and methionine in different media and for different lengths of time. Labeled proteins in the excretory-secretory component and the larval homogenates were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and by 2-dimensional gel electrophoresis. Numerous proteins ranging from 14 to greater than 200 kDa were identified from both the excretory-secretory components and the larval homogenates. Both fractions demonstrated shared and unique proteins. Using timed labeling, age- and stage-specific proteins were identified; at least 2 proteins of approximately 20.5 and 22 kDa were associated in time with the molt from the third to fourth stage. Two proteins of the same molecular weight were specifically recognized by immune dog sera, but not by sera of their infected nonimmune cohorts.     

Purification and Characterization of a O-Methyltransferase Capable of Methylating 2-Hydroxy-3-alkylpyrazine from Vitis vinifera L. (cv. Cabernet Sauvignon)

An S-adenosyl-L-methionine-dependent O-methyl-transferase capable of methylating 2-hydroxy-3-alkyl-pyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatography on DEAE-Sepharose FF, Ether-5PW, hydroxyapatite, G2000SWXL, and DEAE-5PW. The relative molecular mass of the native enzyme estimated on gel permeation chromatography was 85 kDa, and the subunit molecular mass was estimated to be 41 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme also methylates caffeic acid. The V_max for IBHP and caffeic acid were 0.73 and 175 pkatals/mg, respectively, and the respective K_m for IBHP and caffeic acid were 0.30 and 0.032 mM. The optimum pH for IBHP (8.5) was different from that for caffeic acid (7.5).     

Prosomes (proteasomes) of higher plants

From different plant tissues such as tobacco (Nicotiana rustica), potato (Solanum tuberosum), and mung bean (Phaseolus radiatus), ring- or cylinder-shaped particles called prosomes were isolated by either sucrose gradient centrifugation or fast protein liquid chromatography (FPLC). These particles have a diameter of 12 to 14 nm and a length of 16 to 18 nm. They migrate under conditions of nondenaturing gel electrophoresis as one distinct band. Sedimentation coefficient and buoyant density in Cs2SO4 of the plant prosomes were determined by analytical ultracentrifugation to be approximately 23S and 1.23 g/cm3, respectively. The total molecular mass was estimated by gel filtration to be 650 kDa. Plant prosomes are composed of 12 to 15 proteins with molecular masses in the range of 24 to 35 kDa with isoelectric points of pH 5 to 7 as revealed by two-dimensional gel electrophoresis. The protein patterns of prosomes from the three different plant species are very similar. Polyclonal antisera against potato prosomes reacted in Western blots with prosomal proteins of all three plant species. They also bind to some prosomal proteins of animal species. Antisera against animal prosomes react with some proteins of plant prosomes. As shown by lectin blotting, plant prosomes are glycosylated carrying glucosyl- or mannosyl, and N-acetylgalactosaminyl residues. Prosomal preparations contain non-stoichiometric amounts of small RNA of about 80 kDa. These results suggest that plant prosomes are structurally and functionally homologous to prosomes of other eukaryotic cells.     

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: Evidence for intracellular complexes

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with ¹²⁵I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170–180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130–160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with ¹²⁵I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of ¹²⁵I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.     

Comparative studies of thyrotropin releasing hormone diazomethyl ketone and chloromethyl ketone analogs

Diazomethyl ketone and chloromethyl ketone analogs of thyrotropin releasing hormones have been synthesized and studied for their inhibitory effects on thyrotropin releasing hormone-induced release of radioactive ¹²⁵I-labelled hormones from the thyroid gland of eight-week old male Long-Evans rats. When Long-Evans rats were pretreated with thyrotropin releasing hormone diazomethyl ketone (TRH-DMK) or the chloromethyl ketone derivative (TRH-CMK), a dose-related inhibition of thyrotropin releasing hormone-induced ¹²⁵I release was observed which could be partially reversed by thyrotropin stimulating hormone (TSH). The diazomethyl ketone was a more effective inhibitor than the chloromethyl ketone. These compounds may act as an active-site directed antagonists whose effects are unique to the hypothalamo-pituitary-thyroid system.     

Photoaffinity labeling of the calcium channel antagonist receptor in the heart of the cardiomyopathic hamster

The high affinity 1,4-dihydropyridine receptors of the cardiac membrane calcium channel from Syrian Cardiomyopathic hamsters were studied using [3H] PN200-110 and [3H]azidopine as ligands. [3H]Azidopine was photoincorporated covalently into bands of 180, 100, 79, 45 and 31 kDa, as determined by SDS/polyacrylamide gel electrophoresis. Photolabeling of the 180 kDa band is protected by 2 microM [1H]PN200-110 whereas the lower Mr bands are not. Thus, only the 180 kDa band is the calcium channel linked 1,4 dihydropyridine receptor. The photoincorporation into this 180 kDa band is doubled with samples of myopathic hamsters vs. control hamsters. It is suggested that the increase in calcium channel receptors may be involved in the pathogenesis of this cardiomyopathy.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart

The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.     

Structure-activity relationship studies of chloromethyl ketone derivatives for selective human chymase inhibitors

Based on the SAR study of a classical chloromethyl ketone derivative, Z-PheCH2Cl 1, a series of compounds were synthesized. Among all the derivatives, compound 21 was found to be a potent human chymase inhibitor with no inhibitory activity against human leukocyte cathepsin G.     

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.     

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate.

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.     

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1)

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 μM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule-associated protein 1 (MAP-1). The 18 kDa protein copurified with the MAP-1 heavy chains during column chromatography on phosphocellulose, DEAE-cellulose, hydroxyapatite and Bio-Gel A-15m. Incubation of the MAP-1 preparation with a mouse monoclonal antibody to the light chain 1 (LC-1) of MAP-1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP-1 (heavy chains, LC-1, LC-2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP-1. This polypeptide, therefore, is a new light chain (LC-3) of M AP-1.