Structure of two adrenal polypeptides containing multiple enkephalin sequences

Two enkephalin-containing polypeptides of 4000 and 5000 daltons have been isolated from extracts of bovine adrenal medulla. Each polypeptide was purified to homogeneity and subjected to sequence analysis. The entire primary structure of the 4000-dalton polypeptide was established by a combination of automated Edman degradation and chemical analysis of its tryptic peptides. The polypeptide contains two copies of the [Met]-enkephalin sequence, one at the amino terminus and the other at the carboxyl terminus. Chemical analysis of the tryptic peptides and automated Edman degradation of the 5000-dalton polypeptide indicated the presence of a [Leu]enkephalin sequence at the carboxyl terminus and an internal [Met]enkephalin sequence. Both of the above enkephalin-containing polypeptides appear to be intermediates in the biosynthesis of the enkephalins.     

Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster.

Tritiated thymidine was administered at various times, and for various lengths of time, during the larval stages of Drosophila melanogaster. The thymidine was incorporated into DNA and was subsequently detected by autoradiography. These procedures allowed identification of those cells undergoing DNA replication at a particular time and also allowed determination of subsequent changes in relative position of these cells and some of their progeny. An analysis of these data has elucidated characteristic patterns of cell division and cell movement in the formation of the adult nervous system during postembryonic developmental stages.     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

Evidence for suitability of glutathione peroxidase as a protective enzyme: studies of oxidative damage, renaturation, and proteolysis

The stability of glutathione peroxidase was assessed in vitro via oxidative inactivation by peroxides and a peroxidizing fatty acid and by renaturation and proteolysis. The stability of glutathione peroxidase to methyl ethyl ketone peroxide, H2O2, linoleic acid hydroperoxide, and peroxidizing methyl linolenate was compared with the stability of several other enzymes. Sulfhydryl enzymes were the most labile to all four treatments. Some of the enzymes tested were very stable to methyl ethyl ketone peroxide but very labile to linoleic acid hydroperoxide treatment. Glutathione peroxidase in the absence of glutathione was relatively slowly inactivated by each treatment. Linoleic acid hydroperoxide damage to glutathione peroxidase was characterized by release of a nonstoichiometric amount of selenite from the protein. Glutathione peroxidase samples lost all of their activity when (i) acidified to pH 2, (ii) heated 5 min at 100 degrees C, and (iii) treated with 6 M guanidinium hydrochloride or 8.5 M urea and heated 5 min at 100 degrees C. When the pH 2 sample was neutralized or the guanidinium hydrochloride-treated sample was diluted 101-fold, about 80% of the original activity was recovered in 30 min. The samples treated with urea and heat recovered no activity when diluted 101-fold. No loss of glutathione peroxidase occurred during treatment for 24 h within trypsin or thermolysin. Based on these results, glutathione peroxidase appears to be a relatively stable enzyme, and thus is is well-suited to perform its role in peroxide detoxification and prevention of oxidative deterioration of cells.     

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position ?30 and ?65 upstream from the structural genes, starting with a TATA box-like sequence. The 3′ flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.     

Determination of mixtures of benzo[a]pyrene, 2,6-dimethylnaphthalene and their metabolites by high-performance liquid chromatography with fluorescence detection

Nonradiometric techniques were used to identify and quantitate two aromatic hydrocarbons and several of their metabolites in biological samples. High-performance liquid chromatographic separation combined with ultraviolet fluorescence detection provided a sensitive and selective method of trace analysis. Metabolites of benzo[a]pyrene and 2,6-dimethylnaphthalene were prepared by in vitro incubation of these substrates, singly and together, with liver microsomes of coho salmon (Oncorhynchus kisutch). Individual metabolites were separated by high-performance liquid chromatography and quantitated at fluorescence excitation and emission wavelengths characteristic of the corresponding parent hydrocarbon. Future refinement of these techniques may allow the determination of more complex mixtures of xenobiotics and their metabolites in marine organisms.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate.

Resonance Raman spectra of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have been investigated during the reaction of the enzyme with substrate and oxygen. It is found that the spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized by resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm^?1. In the ternary ESO₂ complex, however, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm^?1, respectively. There is no evidence for any dioxygen vibrations in the spectra of ESO₂ complexes prepared with ¹⁶O₂, ¹⁸O₂, and ¹⁶O¹⁸O in the region between 1300 and 200 cm^?1. The results of this resonance Raman study are interpreted to indicate that molecular oxygen is attached only to the substrate (but not iron) in the stable intermediate, and that the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the iron complex. Furthermore, the optical spectrum of the ESO₂ complex (λ_max = 520 nm) is dominated by tyrosinate → Fe(III) charge transfer and contains little or no peroxide → Fe(III) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin.     

Sensitive enzyme assays based on the production of chemiluminescent leaving groups

A new type of synthetic peptide substrate for amidase assay has been devised. The substrates are luminogenic, with potential for extremely high sensitivity, and are here exemplified by Boc- and Z-Ala-Ala-Phe-isoluminol amide. The synthetic substrates were designed to release isoluminol when hydrolyzed by enzyme; isoluminol production was determined by measuring its chemiluminescence. Kinetic constants of the luminogenic substrates were measured with α-chymotrypsin; and levels of the enzyme as low as 50 ng were determined conveniently. A comparison of similar luminogenic, chromogenic, and fluorogenic substrates is presented.     

Circular dichroism and spin-label studies of carp hemoglobin

Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.     

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man₈GlcNAc₂Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

Chloride ion transport and its inhibition in thylakoid membranes

Cl- translocation across energized and nonenergized thylakoid membranes was found to be inhibited by piretanide, an inhibitor of active Cl- transport in fish intestinal epithelia. Piretanide has no effect on photophosphorylation catalyzed by phenazine methosulfate or on Ca2+-dependent ATPase activity of isolated chloroplast coupling factor (CF1). Light-dependent Cl- uptake, contrary to H+ uptake, is severalfold greater at pH 8.0 than at pH 6.7.