Circular dichroism and saccharide-induced conformational transitions of lectins from Ricinus communis.

Conformational studies on lectins from castor beans (Ricinus communis), RCA_I and RCA_II, were performed by using circular dichroism (CD). The CD spectra were similar showing several negative bands at 270–320 nm, a positive region at 230–250 nm, several negative bands at 205–225 nm, and a positive peak at about 195 nm. However, significant differences were observed in the band strength between RCA_I and RCA_II. Lactose, melibiose, and d-fucose induced marked Conformational alterations in RCA_I, whereas weaker effects were produced by d-galactose and l-rhamnose. Saccharide-induced conformational alterations were weaker in RCA_II than in RCA_I, with only lactose and melibiose inducing significant alterations. d-Glucose and 2-acetamido-2-deoxy-d-glucose, which do not inhibit hemagglutination by RCA_I or RCA_II, did not influence lectin conformation. Acetylation of tyrosyl groups with N-acetylimidazole produced changes in the CD bands in the near uv indicating involvement of tyrosine residues. The saccharide effect was most pronounced at 285 nm, a band that was assigned to a tyrosine chromophore. Analysis of the CD bands in the far-uv zone indicated the presence of approximately 50% pleated sheet (β) structure, and 13–15% α-helix in both RCA_I and RCA_II. According to the CD results, the polypeptide chain backbone in the lectins was not affected by the saccharides, whereas significant disorganization occurred in 7 m guanidine-HCl.     

Allosteric transitions and membrane-bound ATPase from rat tissues: The effect of fat deprivation on the allosteric inhibition by fluoride

In rats red a fat-sufficient diet, ATPases (ATP phosphohydrolase, EC 3.6.1.3) from heart, kidney and brain microsomes showed allosteric kinetics for the inhibition by F^?, with values ofn = ?2.0. In rats fed a far-free diet, the values ofn for the ATPases changed from ?2.0 to ?1.0 in heart and kidney microsomes. When these animals were then fed a fat-sufficient diet the values ofn reached the control values. In brain microsomal ATPases no modification of the values ofn were found between both groups of animals. The regulatory properties of the membrane on bound ATPases are discussed.     

The influence of dietary calcium and phosphorus on intestinal calcium transport in rats given vitamin D metabolites.

A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms.In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N^?,N^?-dimethyllysine, N^?,N^?,N^?-trimethyllysine, N^G,N^G-dimethylarginine (or N^G-methylarginine), and N^G,N′^G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N^?-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds.The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N^G,N^G-dimethylarginine (or N^G-methylarginine), and smaller amounts of N^G,N′^G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N^?,N^?,N^?-trimethyllysine and smaller amounts of N^?-methyllysine, N^?,N^?-dimethyllysine, and N^G,N^G-dimethylarginine. These subunits also contained an as yet unidentified methylated compoundThese results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N^?,N^?,N^?-trimethyllysine, with the remainder distributed about equally between N^?-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N^?-methyllysine was apparently in the small acidic proteins, L7 and L12.     

Allethrin interactions with the nicotinic acetylcholine receptor channel

Interactions of the synthetic pyrethroid allethrin with the nicotinic acetylcholine (ACh) receptor/channel were studied in membranes from Torpedo electric organ. Allethrin did not inhibit binding of [3H]ACh to the receptor sites, but inhibited noncompetitively binding of [3H]perhydrohistrionicotoxin ([3H]H12-HTX) to the ionic channel sites in a dose-dependent manner. The inhibition constant (Ki) of [3H]H12-HTX binding in absence of receptor agonist was 30 micro M, while in presence of 100 micro M carbamylcholine it was 4 micro M. This inhibitory effect of allethrin had a negative temperature coefficient. The high affinity binding of allethrin to the channel sites of the nicotinic ACh-receptor may be indicative of a postsynaptic site of action for pyrethroids, in addition to their known action on the sodium channel.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain.

The phases of simple systems involving one type of protein (lysozyme or cytochrome c) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperature than the one observed in the case of the phase with electrostatic interaction.     

Ectodermal interactions during neurogenesis in the glossiphoniid leech Helobdella triserialis

Morphogenetic cell interactions during development were studied by combining cell ablation and cell lineage tracing techniques in embryos of the leech Helobdella triserialis. Ablation of an identified ectodermal teloblast, or teloblast precursor blastomere, on one side of an early embryo was often found to result in the later abnormal migration of the progeny cells of the corresponding contralateral, nonablated teloblast to the ablated side of the embryo; such abnormal migration was termed “midline violation.” Two different kinds of midline violation were observed. Crossover: after ablation of an N teloblast individual stem cell progeny of the contralateral N teloblast sometimes cross the ventral midline of the germinal plate of the embryo. Switching: after ablation of an OPQ teloblast precursor bandlets of stem cells produced by the contralateral O, P, or Q teloblasts sometimes switch to the germinal band of the ablated side at the site of origin of the germinal bands. The occurrence of crossover and switching shows that the eventual site occupied by a progeny cell of a particular teloblast is not automatically determined by its lineage, but also depends on interactions with other cells. Midline violation in the leech embryo CNS does not constitute true regulation, however, since the restoration of neurons to the ablated side is accompanied by a neuron deficit on the nonablated side. The occurrence of the two distinct kinds of midline violation, crossover and switching, may be explained by the relative position of the stem cell bandlets within the germinal bands, and by the geometrical features of the formation of the germinal plate from the germinal bands.     

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.     

Allosteric interactions of the membrane-bound acetylcholine reception: kinetic studies with alpha-bungarotoxin.

The specific and irreversible reaction of a snake neurotoxin, α-bungarotoxin, with the acetylcholine receptor of electroplax membrane preparations from $\text{Electrophorus electricus}$ proceeds by an initial fast phase followed by a slower one. The fraction of the reaction in the fast phase increases with increasing initial toxin concentrations, while the fraction going slowly decreases correspondingly. Both phases are affected by compounds which initiate or inhibit nerve impulse transmissions. The time course of the reaction can be fitted to the sum of two exponentials. The dependence on initial toxin concentration of the two exponentials, and of the fraction of reaction governed by the exponentials, can be fitted to a minimum reaction mechanism which involves at least two types of toxin binding sites with different dissociation constants and ligand-induced conversion of one type of site into the other. The mechanism is consistent with our previous data which showed that activators and inhibitors of membrane electrical potential changes occupy separate sites, only half of which interact. This type of mechanism has been seen in a number of allosteric regulatory enzymes.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

When KCl is added to a solution of G-actin to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot be accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digestibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where actin in the presence of 0.1 m-KCl does not polymerize. This indicates that a confomational change occurs in G-actin before the polymerization step.At sub-critical concentrations in 0.1 m-KCl, actin is in a truly monomeric state as judged by its viscosity characteristics, its inability to enhance the rate of polymerization of G-actin and its possession of ATP as the actin-bound nucleotide. These data support the existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same bound nucleotide as G-actin, but digestion characteristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylated state of the actin-bound nucleotide. Instead, the conformational change appears to be caused by the addition of KCl to G-actin.The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actin monomers form a population of polymerizable molecules which must reach a critical concentration before nucleation and F-actin polymer formation begin.     

Remodeling of sperm chromatin following fertilization: nucleosome repeat length and histone variant transitions in the absence of DNA synthesis

Within the first cell cycle following fertilization the average nucleosomal repeat length of sea urchin male pronuclear chromatin declines by 30-40 base pairs to a value typical of that found in the embryo. This decline occurs after a lag of about 30 min postfertilization, and is accompanied by replication of the male chromatin and accumulation of cleavage-stage (CS) core histone variants. When replication is inhibited by greater than 95% with aphidicolin, the decline in repeat length still occurs, although it is slightly retarded. The decline in repeat length also occurs when protein synthesis is blocked by greater than 98% and DNA synthesis by 60-70% with emetine. The adjustment of nucleosome repeat length therefore can occur in vivo without extensive movement of replication forks across the length of the chromatin, or normal progression of the cell cycle, and appears to require no proteins synthesized postfertilization. Blocking of DNA synthesis or protein synthesis also does not prevent the normal histone variant transitions involved in male pronuclear chromatin remodeling. Although their accumulation is slowed, CS core variants eventually become the predominant male pronuclear histones in their classes when replication is inhibited. Since a shortening of the average nucleosomal repeat length of approximately 10-20% is not sufficient to account for this large acquisition of CS variants, some of the sperm (Sp) core histones are probably displaced from the replication-blocked pronucleus. Therefore, accumulation of CS H2A and CS H2B are temporally correlated with the repeat length transition, whereas replication, normal progression of the cell cycle, and the early histone transitions involving SpH1 and SpH2B are not.     

Effect of complexation of flavin radical with tryptophan on electron transfer rates: a model for flavin-protein interactions

The rates of oxidation of lumiflavin radical by ferricyanide, indole radical and oxygen are decreased by factors of four to ten as a result of complexation with tryptophan. Tyrosine, methionine and glycine were found not to measurably alter the flavin radical reactivity. Similar results were obtained using flavinyl peptides in which tryptophan or methionine were covalently linked to the flavin. These observations suggest that one of the consequences of the interaction between the flavin and a tryptophan side chain in the coenzyme binding site of the flavodoxins is to deactivate the semiquinone form of the enzyme towards oxidizing agents, thereby increasing its stability.     

Hypoxia and cold: influence on cellular oxygen consuming systems in guinea pig liver

Traditionally any biochemical changes found in animals exposed to high altitude have been interpreted solely in relation to hypobaric hypoxia. The present work has been carried out to study the influence of cold and hypoxia in guinea pig native to high altitude.The three major oxygen consuming systems of the liver were measured using cytochrome oxidase as a mitochondrial marker, catalase as a peroxisomal marker, and the o-demethylation of p-nitroanisole and the hydroxilation of hexobarbital as markers for microsomal activity. Serum levels of thyroid hormones (T₃ and T₄) were also determined.Evidence is presented showing that cold produces a dramatic increase of liver catalase and cytochrome oxidase activities, and of serum T₃ and T₄.Interestingly, the increase in thyroid hormones did not precede the increase of the two enzyme activities in the liver of guinea pig exposed to 4°C.On the other hand, it was found that hypoxia appears to have no significant influence upon any of the three major oxygen consuming systems of the liver.     

Influence of alkyl ether chain length of acetyl glyceryl ether phosphorylcholine and its ethanolamine analog on biological activity toward rabbit platelets

The preparation of the potent new lipid chemical mediator, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), through a facile semisynthetic procedure utilizing the vinyl ether-containing lecithins (plasmalogens) of bovine heart muscle as the starting material results in a derivative with mixed chain alkyl residues. In order to focus more closely on the importance of the various components of AGEPC relative to its biological activity, it was of importance to develop a method for isolation of molecules rich in a specific chain length of the alkyl residue. In the current study it was found that a simple thin-layer chromatographic technique, using a solvent system of methanol water (2:1, $\text{v}\text{v}$), afforded an excellent separation of semisynthetic AGEPC into two species, one containing over 95 mol% 16:0 and the other 95 mol% 18:0 alkyl chain species. The same procedure allowed a comparable separation of the species of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylethanolamine (AGEPE) prepared from AGEPC by phospholipase D action. On the basis of their effectiveness in releasing serotonin from washed rabbit platelets, the 16:0-rich derivatives of AGEPC and AGEPE exhibited significantly higher specific activities, from three to six-fold, on a molar basis, respectively, than the corresponding 18:0-rich derivatives. These findings are discussed in relation to the importance of the chain length of the alkyl ether group in expression of the biological activity of AGEPC.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Minor and trace sterols in marine invertebrates : VI. Occurrence and possible origins of sterols possessing unusually short hydrocarbon side chains

Sterols with biosynthetically unusually short side chains (fewer than eight carbon atoms expected for primary squalene cyclization products) have been identified in the extracts of numerous marine invertebrates. The structures of the short side chain and conventional side chain sterols have been determined for various species of Porifera and Coelenterata. Sterol structures were determined by comparison of their mass spectra and gas chromatographic retention times with those of authentic or synthetic samples. Evidence is presented supporting the natural occurrence of these compounds in the tissues of the marine invertebrates as opposed to formation by degradative processes during sample handling or laboratory work-up. The short side chain sterols were found to possess predominantly the androst-5-en-3β-ol nucleus with C-17 alkyl side chains ranging from zero to six carbon atoms. Concentrations of short side chain sterols range from trace levels to over 5% of the sterol mixture in various species. The possible origins of these short side chain sterols are evaluated in the light of current knowledge of sterol function, biosynthesis, dealkylation, microbial degradation, and autoxidation. Known sterol autoxidations are reviewed, and possible singlet oxygen and free radical mechanisms of sterol side chain autoxidation (at physiological temperatures) which may lead to sterols with shortened hydrocarbon side chain are suggested. The possible autoxidative generation of short side chain sterols from known marine sterols by the suggested mechanisms is evaluated through application of the REACT computer program. Predicted short side chains are tabulated for each parent marine sterol side chain and then compared with the compositions of the actual sterols found in the marine extracts examined. The possible natural environmental or in vivo autoxidative formation of the short side chain marine sterols is supported by these evaluations.     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.     

The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti.

The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti. The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.     

The effects of a chitin inhibitor-dimilin- on the production of peritrophic membrane in the locust, Locusta migratoria.

Dimilin, now generally accepted as an inhibitor of chitin production in insects, partially blocks chitin synthesis during the production of the peritrophic membrane. Reduction in chitin leads to reduction in protein in the same proportion. We propose that protein incorporation is affected by the stability of the protein in the matrix such that unbound protein tends to inhibit the addition of further protein.