Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase

Resonance Raman spectroscopy has been employed to investigate the molecular bases for the markedly different properties of horseradish oxyperoxidase and oxymyoglobin. The porphyrin core of oxyperoxidase is slightly more expanded with the iron atom closer to the porphyrin plane, and there is greater iron d pi-to-oxygen pi backbonding compared to oxymyoglobin. The iron-oxygen (stretching or bending) bands are observed at 570 and 562 cm-1, respectively, for oxymyoglobin and oxyperoxidase, and the iron-His stretching bands have been tentatively identified at 276 and 289 cm-1, respectively. It is suggested that the stronger iron-His bond in oxyperoxidase facilitates greater iron d pi-to-oxygen pi backdonation by raising the energy of the iron d pi orbitals closer to the energy of the oxygen pi orbitals. This weakens the O-O bond and activates dioxygen for use as an electron acceptor in the peroxidase-oxidase reaction.     

The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli

Interaction of Phe-tRNA.elongation factor Tu.GTP with poly(U)-programmed ribosomes containing an occupied P site can be described by a three-step kinetic mechanism. Initial binding is followed by the cleavage of GTP, and then a new peptide bond is formed. Rate constants controlling the first and third of these reactions are known, but only a lower limit for the rate constant of the cleavage step has been reported. We have determined this rate constant to be 20 s-1 at 5 degrees C, 30 s-1 at 15 degrees C, and 50 s-1 at 25 degrees C. This is much faster than the reverse step of the initial binding process and implies that the intrinsic accuracy of the ribosome in the initial selection step is sacrificed in favor of speed. The similarity of the kinetic and chemical mechanism of this GTP cleavage step with other nucleoside 5'-triphosphatases is discussed.     

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.     

A second gene in a Balbiani ring. Chironomus salivary glands contain a 6.5-kb poly(A)+ RNA that is transcribed from a hierarchy of tandem repeated sequences in Balbiani ring 1

A second gene has been discovered at a previously studied Balbiani ring in Chironomus. Northern hybridizations demonstrated that cDNA clone pCt35 originated from a salivary gland specific 6.5-kilobase (kb) RNA that was abundant, nonribosomal, and apparently poly(A)+. pCt35 had a 120-base pair (bp) insert with 1.6 copies of a 75-bp sequence that contained two open reading frames. Southern hybridizations indicated that pCt35 was homologous to at least a 4-kb block of genomic DNA organized as a hierarchy of 150- and 300-bp tandem repeats. In situ hybridization localized these sequences to Balbiani ring 1. From these results we postulated that a 6.5-kb RNA gene may have evolved by stepwise duplication and amplification of a 75-bp ancestral sequence.     

Analysis of the direct effects of prostaglandins on human sperm function

Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between individual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.     

Myxosporean infections in cultured tilapias in Israel

Five new species of myxosporean parasite are described from cultured tilapias in Israel. These are: Myxosoma sarigi, Myxosoma equatorialis, Myxobolus israelensis, Myxobolus agolus, and Myxobolus galilaeus. The first four were found in hybrids of Oreochromis aureus X Oreochromis niloticus while Myxobolus galilaeus was found in Sarotherodon galilaeus. In addition, M. sarigi, M. israelensis, and Myxobolus sp. were also found in S. galilaeus. In the light of the present study, the taxonomy of myxosporean infections in tilapias is modified. Mature spores may localize in the melano-macrophage centers of the spleen and kidney where they may eventually be destroyed. No cases of mortality have so far been associated with these parasites.     

Innervation of the limb accessory flexor muscle in several decapod crustaceans. II. Electrophysiology

The innervation of the distal and proximal heads of the accessory flexor muscle in three portunid crabs and two non-portunid decapods was studied electrophysiologically. In all species studied, the proximal head received only the two previously reported accessory flexor axons, an excitor and an inhibitor. The same two axons also innervated the distal head in all species, but in the portunids the distal head also received excitation from at least three, and probably sometimes four, of the main flexor excitor efferents. The accessory inhibitor exerted very strong effects in the tonic muscle fibers found in the proximal head and in the most proximal bundle of the distal head. The newly described inhibitory and excitatory distributions may have important implications for locomotory behavior.     

Relationship between progesterone receptor binding and progestin biological activity

The binding affinities of a series of steroidal compounds for the hamster uterine progesterone receptor were determined using two sets of incubation conditions. These competitive binding conditions were designed to deduce the relative rates of ligand dissociation from the progesterone receptor. The progestin activity of these compounds was also determined in a bioassay employing the measurement of diamine oxidase in the traumatized hamster uterus. Steroids could be classified into two categories based on either an increase or decrease in relative binding affinity (RBA) with increasing time of competitive incubation. The mean (+/- SEM) progestin biopotency for the compounds having an increase in RBA was 120 +/- 18 (progesterone = 100), while the biopotency for compounds having a decrease in RBA was only 44 +/- 17. This difference was significant (P less than 0.01). Linear regression analyses revealed significant correlations between the RBAs and progestin biopotencies. Compounds showing a decrease in RBA with increasing time of incubation did not have antiprogestin activity. Kinetic studies of this type should be useful for selecting compounds with potent agonistic activity, but cannot unequivocally predict antihormonal activity.     

FSH stimulation of cytosolic protein synthesis in cultured pig Sertoli cells

The effects of FSH on the cytosolic protein synthesis by a primary culture of immature porcine Sertoli cells were studied. Cells were cultured in a chemically-defined medium for 3 days and, on day 3, they were incubated for 40 h in another medium containing labelled amino-acids either in the presence or absence of 50 ng/ml porcine FSH (pFSH). In control cells, about 107 spots (pI in the range of 5 to 8) were identified by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled cytosolic proteins. pFSH treatment produced a marked increase of seven proteins whose molecular weights and isoelectric points are respectively comprised between 25 to 58 K and 5.5 to 5.8. In addition, pFSH treatment induced a slight but constant increase of two other proteins (mol. wt: 24 and 12 K and isoelectric point: 5.3).