Indomethacin, salicyclates and prostaglandin binding

Aspirin, salicyclic acid and indomethacin reversibly inhibit prostaglandin binding to human serum proteins. This effect was demonstrated in the sera of normal subjects and of rheumatoid arthritis patients treated with aspirin as well as by addition of these drugs to serum . The displacement of serum prostaglandins by salicylate is likely to affect the kinetics of prostaglandin transport and may facilitate the delivery of prostaglandins from serum to tissue receptors.     

A new method for rapid determination of sperm concentration in bull and ram semen

A simple method for rapid evaluation of bull and ram sperm concentration is described. In this technique, a sample from an undiluted specimen was placed in a special 10-mum counting chamber and examined either by an ordinary or a phase-contrast microscope. The pattern distribution of the observed spermatozoa was matched as closely as possible with one of five pictures of a standard scale. This scale was prepared from serial photomicrographs that ranged from 100 to 1500 million per ml. The number that appeared at the corresponding photograph immediately indicated the sperm concentration of the tested specimen. In this way values up to 1500 million per ml could be determined rapidly with no further procedures. More concentrated specimens needed slight dilution to make them suitable for direct evaluation. Accuracy and reliability were statistically evaluated; the mean deviation from the conventional method was less than 15.2% with confidence level of 95%.     

A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. 31P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO2, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO2 efflux. Most of the acid, however, cannot be attributed to CO2 release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO2 and protons from the cytoplasm into the surrounding seawater.     

Diabetes-induced variation in hepatic binding protein

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozotocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrificed 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70% of the normal level.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Studies on the specificity of sperm binding in echinoderm fertilization

Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus, we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata.     

Interactions of heme proteins with hydrogen peroxide: protein crosslinking and covalent binding of benzo[a]pyrene and 17 beta-estradiol

This work reveals two biochemical effects of hydrogen peroxide treatment on hemoglobin, myoglobin, and cytochrome c. First, these heme proteins rapidly formed covalently crosslinked dimers and polymers detectable by detergent gel electrophoresis. Second, when treated in the presence of radioactive benzo[a]pyrene or 17 beta-estradiol, the heme proteins became covalently labeled. Nonheme proteins exhibited both cross-linking and radioactive labeling upon peroxide treatment in the presence but not the absence of heme protein or free hemin. Benzoyl peroxide or glucose and glucose oxidase effectively replaced direct addition of hydrogen peroxide. These results indicate that adventitious peroxidase activity expressed by oxygen carrying and electron transport proteins yields active oxygen species that can damage these heme proteins and nearby macromolecules, a possible biochemical mechanism for the lethal and other deleterious intracellular effects of peroxide.     

A heme binding site on myelin basic protein: characterization, location, and significance

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.     

Fatty acid synthetase, malic enzyme and other NADP+ binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.     

Cu(II) binding by substituted 1, 3, 5-triazine herbicides

s-Triazine herbicides form relatively stable complexes with cupric ions, complexes in which the major binding site was found to be a cyclic nitrogen N5. Polarographic data have shown that cuprous ions also form quite stable complexes with the studied ligands. The stability constants strongly suggest that s-triazines act in solutions as the monodentate ligands, a result which agrees well with earlier X-ray results.     

The A protomer of islet-activating protein,pertussis toxin,as an active peptide catalyzing ADP-ribosylation of a membrane protein

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A protomer and a B oligomer. IAP and its A protomer were equipotent, on a molar basis, in enhancing GTP-dependent adenylate cyclase activity and in causing ADP-ribosylation of the 41,000 M_r protein when directly added to the cell-free membrane preparation from rat C6 glioma cells. Similar actions of IAP observed upon its addition to intact C6 cells were not mimicked by its A protomer, indicating that the A protomer had to be associated with the B oligomer to become accessible to its site of action on the inner surface of the membrane of intact cells. The A protomer, but not IAP, exhibited NAD-glycohydrolase activity in the reaction mixture lacking cellular components but containing dithiothreitol. Their actions on membranes were not accelerated by dithiothreitol, but markedly suppressed by oxidized glutathione. Thus, C6 cell membranes may possess certain “processing” enzyme(s) responsible for releasing the A protomer from the IAP molecule and for reductive cleavage of an intrachain disulfide bond in the released protomer, thereby producing an active peptide which functions to cause ADP-ribosylation of one of the subunits of guanine nucleotide regulatory protein in the receptor-adenylate cyclase system.     

Motility activation, respiratory stimulation, and alteration of Ca2+ transport in bovine sperm treated with amine local anesthetics and calcium transport antagonists

Optimal concentrations of dibucaine and other structurally related tertiary amines, variously classified as local anesthetics, Ca²⁺ transport antagonists or calmodulin-directed agents greatly stimulate respiration and the motility of bovine spermatozoa in a reversible manner. Because dibucaine also increases lactate production by sperm made dependent upon glycolysis, the induced metabolic stimulation is probably a secondary response to the greater energy demands resulting from increased motility. Microscopic and time lapse photomicrographic examinations indicate that dibucaine increases the proportion of motile cells and alters the predominant linear swimming path to a peculiar figure eight pattern of movement. Frame by frame analysis of video recordings indicate that this pattern of movement closely resembles, or is identical to the characteristic “motility activation” that occurs during the capacitation sequence which obligatorily precedes fertilization of many, if not all, mammalian species. Dibucaine and the Ca²⁺ transport antagonists, D600 and TMB-8, inhibit the net uptake of Ca²⁺ by sperm suspensions. The dose-response relationships indicate that inhibition of Ca²⁺ uptake does not bear a causal relationship to the activation of motility and metabolism and further suggest a common action of these agents rather than selective effects of D600 and TMB-8 upon Ca²⁺ channels in the sperm plasma membrane. In addition, dibucaine and D600 each induce release of that Ca²⁺ which was accumulated by intact sperm in a preliminary incubation in the absence of the drugs and also inhibit uptake of Ca²⁺ by digitonin-treated sperm. Apparently, therefore, local anesthetics have a direct deleterious action on sperm mitochondrial function. Treatment with the high concentrations of local anesthetics that are required to inhibit uptake of Ca²⁺ completely results in a rapid and irreversible immobilization of the sperm. This loss of motility is either not mediated, or mediated indirectly, through an action of the drug on mitochondrial function because sperm similarly become immotile when a glycolytic substrate is supplied simultaneously.     

Antibodies to muscarinic acetylcholine receptors in myasthenia gravis

IgG obtained from patients with myasthenia gravis block the specific binding of the muscarinic antagonists (³H)-N-methyl-4-piperidyl benzilate (4NMPB) and (³H)-Quinuclidinyl benzilate to rat brain muscarinic acetylcholine receptors. IgG obtained from healthy controls have a much smaller effect. The inhibitory effect of the myasthenic IgG increases linearly with immunoglobulin concentration and has no effect on the affinity of the muscarinic receptors towards (³H)-4NMPB (K_D = 0.7 ± 0.1 nM). This implies that the inhibition is probably due to the blocking of some of the muscarinic receptors by the myasthenic IgG, and not due to alteration in affinity of all the receptors. it remains to be established whether the presence of antimuscarinic receptor activity in the serum of myasthenic patients is of importance in the pathophysiology and diagnosis of myasthenia gravis.     

Precocene II-induced alate production in isolated and crowded alate and apterous virginoparae of the aphid, Macrosiphum euphorbiae

Precocene II was applied at doses ranging from 0.05 to 0.70 μg per individual to newly moulted adult alate and apterous virginoparae of Macrosiphum euphorbiae kept isolated or in groups of 10 per plant, under an 18L:6D photoperiodic regime. While isolated controls of both morphs produced exclusively apterous progeny, alatae virginoparae were produced in generally dose-dependent proportions by precocene-treated individuals. Grouped controls of both morphs produced alatiform progeny as expected, but in precocene—treated groups, the proportions of alate progeny generally increased as a function of dose. The overall proportions of alate offspring produced, and numbers of days after treatment when morph production was affected, were generally greater for alatae than for apterae, indicating a greater sensitivity to precocene in alatae. However during the first few days after treatment, the alatizing effect of precocene was stronger for apterae, suggesting that the first embryos produced by alatae were irreversibly determined as apterae.In an experiment where isolated alatae and apterae received 0.5 μg of precocene II at different ages ranging from 1 to 13 days after the adult moult, the alatizing effect of the compound, measured by the persistence of alate production, varied with age and morph. While in alatae, the persistence decreased more or less regularly with age, in apterae it initially increased to a maximum in the middle of reproductive life, and subsequently decreased. The results provide support for the hypothesis that juvenile hormone is involved in regulating alary dimorphism in M. euphorbiae.     

Adrenocortical cyclic GMP-dependent protein kinase: purification, characterization, and modification of its activity by calmodulin, and its relationship with steroidogenesis

Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the M_r of the enzyme was 145,000. This protein was composed to two identical subunits each with M_r of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N²-monobutyryl cyclic GMP > N², O²-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis.     

An acidic protein which assembles nucleosomes in vitro is the most abundant protein in Xenopus oocyte nuclei

Eggs and oocytes of the frog Xenopus laevis contain an acidic thermostable protein which promotes assembly of nucleosomes from histones and DNA in vitro (Laskey et al., 1978). Analysis of the abundance and intracellular distribution of this protein reveal that it is exclusively localized within the oocyte nucleus where it is the most abundant protein. Its intranuclear concentration is 5 to 7 mg/ml representing 7 to 10% of the total nuclear protein. Micro-injection into oocyte cytoplasm demonstrates that the property of intranuclear migration resides in the mature protein. The injected protein migrates into the nucleus efficiently and becomes distributed throughout the nucleoplasm but it does not associate preferentially with structures containing DNA.