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PubMed1982

Measurement of receptor-independent lipoprotein catabolism using 1,2 cyclohexanedione-modified low density lipoprotein.

The utility of 1,2 cyclohexanedione-modified low density lipoprotein (CHD-LDL) as a marker for the measurement of receptor independent LDL catabolism has been assessed by examining its metabolic properties in cultured human fibroblasts and in rabbits. Cell culture studies showed that the inhibition of high affinity membrane receptor binding produced by the modification could be partially reversed by prolonged incubation of the CHD-LDL at 37 degrees C. Pre-exposure of the complex to alkaline pH (pH 10.5) prevented this and yielded a product that was apparently stable. Despite its regained ability to bind to the fibroblast receptor, 125I-labeled CHD-LDL incubated at 37 degrees C for 24 hr either in vivo or in vitro was removed from rabbit plasma in the same manner as freshly prepared 131I-labeled CHD-LDL and as 131I-labeled CHD-LDL that had been treated at pH 10.5. However, its plasma clearance was significantly faster than that of reductively methylated LDL. We believe that this may result from differential catabolism of these modified lipoproteins rather than from susceptibility of the CHD-LDL to receptor-directed catabolism.

Animals↗
PubMed1983

Botulinum neurotoxin type E: studies on mechanism of action and on structure-activity relationships.

Single chain type E botulinum neurotoxin was isolated from culture fluids of Clostridium botulinum (strain Alaska E-43). The neurotoxin, which migrated as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate, had a molecular weight of approximately 147,000. Single chain type E neurotoxin that was exposed to trypsin was converted to a dichain molecule. Pretreatment of the single chain molecule with 1,2-cyclohexanedione, a reagent that selectively modifies arginine residues, inhibited trypsin-induced generation of the dichain molecule. In dose-response experiments (10(-13) to 10(-9) M) on the isolated neuromuscular junction (phrenic nerve-hemidiaphragm preparation), the dichain neurotoxin was approximately two orders of magnitude more potent than the single chain neurotoxin. Neither specie of neurotoxin (1 pmol/mouse, in vivo; 1 X 10(-11) M, in vitro) was very effective in blocking autonomic transmission (vagus nerve-atrium preparation). The neuromuscular blocking action of the dichain molecule was divided into a sequence of three steps. There was an initial binding step that was relatively rapid, little influenced by temperature and which left the neurotoxin partially accessible to the neutralizing effects of antitoxin. There was a translocation step that was temperature dependent, antagonized by ammonium chloride and methylamine hydrochloride and which caused the neurotoxin to become inaccessible to the neutralizing effects of antitoxin. Finally, there was an intracellular lytic step, during which the toxin blocked excitation-secretion coupling.

Animals↗
PubMed1984

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase.

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.

Animals↗
PubMed1980

Reversible modification of arginine residues in neocarzinostatin. Isolation of a biologically active 89-residue fragment from the tryptic hydrolysate.

Reaction of the antitumor protein neocarzinostatin with 1,2-cyclohexanedione in 0.25 M borate buffer, pH 9.0, resulted in complete modification of arginine residues in positions 66, 67, and 78. The arginine-modified protein lost its native structure and was biologically inactive in the inhibition of growth of HeLa cells, inhibition of DNA synthesis, and in vitro DNA strand scissions. Trypsin hydrolysis of 1,2-cyclohexanedione-modified neocarzinostatin resulted in selective cleavage of the Lys-Val (positions 20 and 21) bond of the primary structure yielding NH2-terminal 1-20 and the COOH-terminal 21-109 residue fragments. The latter contained modified arginine residues. Both peptide fragments were biologically inactive. Treatment of the arginine-modified neocarzinostatin and the arginine-protected 89-residue fragment with 0.25 M Tris-acetate buffer, pH 9.0, for 15 h resulted in the release of 1,2-cyclohexanedione, regenerating all three arginine residues. The regenerated protein and the 89-residue fragment were fully active biologically. Further, the regenerated 89-residue fragment possessed 70% of the reactivity of neocarzinostatin with antibody raised against the native protein. The conformation of the 89-residue fragment was almost identical with that of the native protein in CD spectral properties.

Amino Acids↗
PubMed1983

The sites of neurotoxicity in alpha-cobratoxin.

We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the acetylcholine receptor protein from Torpedo marmorata. (i) The toxin derivatives which were fully modified at lysine (penta-epsilon-N,N-dimethyl lysine; penta-epsilon-N-acetyl lysine), arginine (penta-N7,N8-(1,2-dihydroxycyclohex-1,2-ylene arginine), and tyrosine (mononitrotyrosine) all had significant remaining toxicity and affinity of binding. (ii) The "extra" disulfide of alpha-cobratoxin was selectively reduced and alkylated. Depending on the charge, size, and hydrophobicity of the attached groups, derivatives were obtained that bound to the acetylcholine receptor with higher (di-S-carboxyamidomethyl), about equal (di-S-pyridylethyl), or lower (di-iodoacetaminoethylnaphthylamine-5-sulfonic acid) affinity than the unmodified toxin. (iii) A fully reduced and carbamidomethylated derivative of alpha-cobratoxin obtained by repeating the procedure for selective reduction six times still bound with appreciable affinity (KD approximately 3 X 10(-6) M) to the acetylcholine receptor. We conclude that neither a single positively charged residue nor tyrosine nor the integrity of the disulfides is absolutely essential for toxicity. Furthermore, the single tyrosine and the area around the extra disulfide do not participate in the binding to the receptor. Together with previous findings on this interaction, this suggests a multipoint attachment of toxin and receptor involving several locally separate structural elements of the toxin.

Acylation↗
PubMed1984

Arginine modification in elastase. Effect on catalytic activity and conformation of the calcium-binding site.

Chemical modification of 2 +/- 0.5 arginine residues of porcine pancreatic elastase by 1,2-cyclohexanedione leads to an 85 +/- 5% loss of activity with the specific substrate N-succinyltrialanine p-nitroanilide. Modification of additional arginines does not completely abolish the enzyme activity. The modification reaction is very fast (second order rate constant = 0.24 M-1 S-1) and involves only arginine residues. Acetyltetraalanine or trifluoroacetyltetraalanine decreases the rate of cyclohexanedione-induced inactivation of the enzyme but does not significantly change the number of modified arginine residues. Other dicarbonyl reagents, butanedione or phenylglyoxal, also react with elastase but at much lower rates. Cyclohexanedione-modified elastase is partially active against a series of synthetic substrates of varying chain length. The partial inhibition results from a 2- to 5-fold increase in Km while kappa cat is increased for most substrates. For N-succinyltrialanine p-nitroanilide both the acylation and deacylation rate constants are decreased. The Ki values of a series of acetylated and trifluoroacetylated inhibitors increase 2- to 5-fold. Modified elastase is still able to react with fibrous elastin and with plasma alpha 1-proteinase inhibitor but at significantly lower rates. Modification of one arginine residue alters the properties of the calcium-binding site of elastase as demonstrated by terbium luminescence experiments. The affinity of enzyme for terbium is decreased by a factor of 10 and the circularly polarized luminescence spectrum of the terbium-elastase complex is considerably flattened. Modification of further arginine residues does not increase the extent of these alterations. Circular dichroism shows that the overall conformation of elastase is not altered following arginine modification. We speculate that the two residues modified by cyclohexanedione are Arg 65, located at about 8 A from the metal ion-binding site, and Arg 217A, located at the S'3 subsite of elastase.

Animals↗
PubMed1978

[Study of the role of arginine residues in aspartate transaminase from chicken heart cytosol].

Reaction of 1,2-cyclohexanedione with chicken heart cytosolic aspartate transaminase results in loss of enzyme activity complying to first order kinetics up to 70% inactivation. The inactivation rate is markedly decreased in the presence of alpha-ketoglutarate, glutarate or alpha-methylaspartate. The number of arginine residues modified per subunit was approximately two (in enzyme preparations which retained 30% residual activity). The diketone-modified enzyme nearly completely loses affinity for alpha-methylaspartate and glutarate; in contrast, its ability to bind alpha-alanine and catalyze its transamination half-reaction with the bound coenzyme remains unimpaired. From these data it can be inferred that a functional arginine residue is the cationic binding site for the distal carboxyl group of the substrates. The transaminase apoenzyme was inactivated with cyclohexanedione at the same rate as reconstituted holoenzyme. Measurements of circular dichroism showed that the modified apoenzyme is capable to bind pyridoxal-P. No evidence was obtained for the presence of an arginine residue in the coenzyme binding site.

Alanine↗
PubMed1983

The role of arginyl residues in estrogen receptor activation and transformation.

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

A steady state kinetic analysis of the reaction of chloroperoxidase with peracetic acid, chloride, and 2-chlorodimedone.

The chloroperoxidase-peracetic acid-chloride-2-chlorodimedone system is used as a model for the investigation of enzymatic halogenation reactions. Systematic variation of the concentrations of the three substrates under steady state conditions yields sets of kinetic parameters containing both kinetic and mechanistic information. Three distinct enzyme species are involved in the halogenation cycle: native enzyme, compound I, and a short-lived halogenating intermediate. Analysis of the kinetic data is complicated by the fact that chloride serves as a substrate in the second step and as an inhibitor of the first step of the overall halogenation reaction. The inhibitor binding site on the native enzyme must be protonated prior to the binding of chloride. Chloride appears to be a competitive inhibitor for both compound I formation and cyanide binding to chloroperoxidase. Only the latter reaction can be studied directly in stopped-flow experiments since compound I disappears rapidly by reacting with chloride present in solution. Rate constants are calculated for the individual steps of the reaction at four different pH values. The rate constant for compound I formation is independent of pH, but the actual rate is reduced at lower pH values due to pH dependence of the chloride inhibition. The rate constant for the oxidation of chloride by compound I decreases with increasing pH. The rate of the halogenation step appears to be independent of pH.

Acetates↗
PubMed1983

[Synthesis and antitumor properties of carminomycin 13-cyclohexylidenhydrazone].

Carminomycin 13-cyclohexylidenhydrazone (CCH) was prepared by interaction of carminomycin 13-hydrazone with cyclohexane. The antiblastomic properties of CCH were studied on mice with transplantable tumors. The preparation was administered intravenously or orally. The studies showed a high antitumor activity of CCH. When CCH was administered intravenously to mice with lymphosarcoma LIO-1, the antitumor effect selectivity of it was practically equal to that of carminomycin. When used in doses equivalent by their toxicity to those of carminomycin, CCH had practically the same inhibitory effect on sarcoma 180 as carminomycin. When used orally in doses equivalent by their toxicity to those of carminomycin, CCH was more effective than carminomycin in treatment of mice with lymphosarcoma LIO-1, sarcoma 180 and lymphadenosis NK/Ly.

Administration, Oral↗
PubMed1981

Chemical modification of critical catalytic residues of lysine, arginine, and tryptophan in human glucose phosphate isomerase.

Human glucose phosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. A specific lysine was found to stoichiometrically react with pyridoxal 5'-phosphate forming a reversible Schiff base which could be reduced with NaBH4. The covalently modified enzyme was specifically cleaved with hydroxylamine at three labile Asn-Gly sequences yielding a series of peptides which were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis. The modified lysine was located in the COOH-terminal peptide. A critical arginine residue/subunit was found to be stoichiometrically modified with either 2,3-butadione or cyclohexadione. At high concentrations of butadione, an irreversible nonspecific modification of essentially all arginines occurred. An essential tryptophan residue was found to be stoichiometrically modified with N-bromosuccinimide in a similar fashion. Each of the chemical modifications of these three residues followed pseudo-first order and rate saturation kinetics and the modifications were prevented by the presence of substrates or competitive inhibitors. Circular dichroic spectral studies and analytical gel filtration indicated that these modifications have no effect on the quarternary structure and little effect on the secondary and tertiary structures of the enzyme. However, the extensive modification of arginine with butadione caused a dissociation of the enzyme into monomers and significant changes in tertiary structure. These studies provide new insights into functional aspects of isomerization and also provide an effective method for evaluating structural consequences of chemical or genetic modification of the enzyme.

Affinity Labels↗
PubMed1981

[Structure and conformation-activity relationships of cyclic acetylcholine analogues / 12th communication: Synthesis and cholinergic properties of stereoisomeric 3-acetoxythiacyclohexanes (author's transl)].

In the course of investigations of structure and conformation-activity relationships of cyclic acetylcholine analogues, both the enantiomers of trans-3-acetoxy-1-methylthioniacyclohexane were prepared. These two esters and the corresponding racemate of the cis-ester were tested for nicotine- and muscarine-like activity. The stereoisomeric cyclic analogues differ substantially in pharmacological activity. The cis-sulfonium ester shows the highest nicotinic potency (1/25 the nicotinic potency of acetylcholine), and the (+)-trans-ester has no agonistic properties when tested at nicotinic receptors, but it shows the highest muscarinic potency in this series.

Acetylcholine↗