Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.     

Switch in pattern formation after puncturing the anterior pole of Smittia eggs (Chironomidae, Diptera).

The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region.     

Leaf production and nitrogen and phosphorus tissue content of Littorella uniflora (L.) Aschers. In relation to nitrogen and phosphorus enrichment of the sediment in oligotrophic Lake Hampen,Denmark

Littoral sediments with a dense population of Littorella uniflora (L.) Ascers. were artificially enriched with nitrogen and phosphorus. Responses to increasing levels of nutrients were recorded as leaf production and tissue nutrient content. Phosphorus enrichment resulted in increasing P concentrations in all plant fractions (leaves, stem and roots), whereas only leaves and roots showed increasing N concentrations when inorganic N was added. Leaf production increased significantly after increasing the sediment P level 2, 3 and 5 times. Nitrogen did not affect leaf production. The tissue content of total N and P in control plants averaged 3.29 and 0.28%, respectively. Thus the critical P level in L. uniflora seems to be about 0.28%, which is higher than previously published values. It was also found that L. uniflora translocates phosphorus from old to younger leaves.     

Purification, characterization, and properties of beta-glucosidase enzymes from Sclerotium rolfsii

Four β-glucosidase enzymes were extensively purified from the culture filtrates of Sclerotium rolfsii and some of their physicochemical properties studied. All the enzymes showed a single protein band in sodium dodecyl sulfate-gel electrophoresis and in disc gel electrophoresis at pH 8.9 and 4.3. The purified β-glucosidases were free of endoglucanase (carboxymethyl cellulose viscosity-lowering activity). All the enzymes are glycoproteins and are composed of one polypeptide chain. The molecular weight of the four β-glucosidases varies between 90,000 and 107,000. The pH and temperature optima of the four β-glucosidases are 4.2 and 68 °C with p-nitrophenyl-β-d-glucoside and 4.5 and 65 °C with cellobiose as substrate. The isoelectric points for the enzymes are 4.10, 4.55, 5.10, and 5.55, respectively. The specific activities of the enzymes with cellobiose as substrate are 55, 78, 175, and 51 μmol glucose released per minute per milligram protein, respectively. The enzymes are inhibited by the reaction product glucose, and by glucono-δ-lactone and nojirimycin. A carboxylate group is implicated in the catalysis of β-glucosidase.     

Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

Recent developments in the activation process of bovine chymotrypsinogen A

A brief account is given of the history, distribution, and activation events of proteins of the bovine chymotrypsinogen family. Recent developments in the investigation of the activation process of bovine chymotrypsinogen A are discussed, and a revised scheme for the overall activation process is presented.     

Developmental studies of lethality associated with the antennapedia gene complex in Drosophila melanogaster

A number of dominant homoeotic mutations are localized to the proximal right arm of chromosome 3 of Drosophila melanogaster and are thought to represent members of a gene complex that controls normal determinative decisions in the head and thorax. We have designated this complex the Antennapedia gene complex (ANT-C). Developmental studies were done to investigate the nature of the lethality associated with members of two of the complementation groups within ANT-C. The first complementation group, represented by the mutant Multiple Sex Combs (Msc) is characterized by embryonic lethality when heterozygous with a deletion of the ANT-C. The second complementation group consists of Antennapedia (Antp), Antennapedia-Extra Sex Combs (Antp^Scx), and the lethals recovered as revertants of Antp^Ns. When heterozygous for a deletion of the ANT-C or in heterozygous condition with each other, the members of this group show effective lethal phases spanning from embryo-larval boundary to late larval stages. Wakimoto and Kaufman (1981) show that the Antp⁺ gene acts to establish normal determinative states in the thorax. In the present work, transplantation of eye-antennal disks from lethal individuals heterozygous for two different Antp^Ns revertant chromosomes into wild-type hosts allowed the assessment of the function of the Antp⁺ allele in the antenna. Since these transplants formed only antennal structures and showed no evidence of the antennal → leg transformation seen in Antp^Ns controls, we conclude that the wild-type function of the Antp locus is not necessary for the establishment and/or maintenance of the antennal determined state. We suggest that regulatory mechanisms associated with the Antp⁺ structural gene normally function both to allow its expression in the thorax and to repress it in the antenna.     

Effects of streptozotocin in the male guinea pig: a potential animal model for studying diabetes

The effects of acutely administered streptozotocin in the male guinea pig were studied for a period of 18 days following treatment. A single intracardiac injection of streptozotocin (150 mg/kg) was administered on Day 0. On Day 2, plasma glucose concentrations were not significantly different from control levels. On Day 7 and 18, an oral glucose tolerance test was performed with streptozotocin-treated animals receiving an acute injection of either insulin (18 U/kg, i.m.) or saline 90 minutes prior to glucose loading. On Day 7, streptozotocin-treated animals receiving saline had significantly elevated plasma and urine glucose concentrations at 3 hours after glucose loading when compared to controls. Streptozotocin-treated animals receiving insulin however, had significantly lower plasma glucose concentrations at 3 hours while urinary glucose was equal to control values. The second glucose tolerance test performed on Day 18 yielded similar results. Necropsies were performed on animals that died after Day 6. Lesions found in the streptozotocin-treated animals included: small and irregular pancreatic islets, pyknotic nuclei and degranulation of beta cells, renal proximal tubule swelling and vacuolization, adrenal cortical hyperplasia, hepatocyte vacuolization, and visceral fat atrophy. Animals surviving until Day 18 were sacrificed and found to have significantly elevated kidney and adrenal weights compared to controls. These changes illustrate the effectiveness of streptozotocin in the acute chemical induction of diabetes in an animal model (guinea pig) which, like humans, requires a dietary source of ascorbic acid.     

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.     

Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein

A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing. In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing. Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease. The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis. The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length. Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP. RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981). Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III. Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein. These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP. The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.     

Defects in embryogenesis in mutants associated with the antennapedia gene complex of Drosophila melanogaster

Embryogenesis in individuals with mutations or deficiencies of the genes in the polytene interval 84A-84B1,2 of Drosophila melanogaster was examined using scanning electron microscopy (SEM). The developmental function of this region of chromosome 3 is of particular interest since it contains the Antennapedia Gene Complex (ANT-C), a gene cluster that includes the homoeotic proboscipedia (pb), Sex combs reduced (Scr), and Antennapedia (Antp) loci. The results of SEM studies, clonal analyses, and temperature-shift experiments show that the fushi tarazu (ftz) and zerknullt (zen) genes, which map between pb and Scr, are involved in processes initiated during embryogenesis. The activity of ftz+ appears to be required within the first 4 hr of development for the establishment of the proper number of segments in the embryonic germ band. Individuals with ftz mutations or deficiencies produce only half the normal number of segments. Each of the segments is twice the normal width and is apparently comprised of cells that would normally form two separate metameres. The zen allele is required from about 2-4 hr of embryogenesis. Mutations of this gene result in disturbances of morphogenetic movements during gastrulation. The mutant phenotype is characterized by the absence of the optic lobe, defects in involution of the head segments, and in some cases, failure of germ band elongation. A requirement during embryogenesis for the activities of other genes residing in the 84A-84B1,2 polytene interval is suggested by the phenotypes of individuals heterozygous or homozygous for chromosomal deficiencies. Using the deficiencies Df(3R)AntpNs+R17, Df(3R)Scr, and Df(3R)ScxW+RX2, we examined the effects of deleting the distal portions or all of the 84A-84B1,2 interval. The defects in deletion heterozygotes suggest that the wild-type activity of some gene(s) other than zen, within or just adjacent to the 84B1,2 doublet, is required to complete normal head involution. The deletion of all the loci in the 84A5-84B1,2 interval results in grossly abnormal morphology and morphogenesis of the gnathocephalic appendages of the embryo. From these studies we conclude that mutations and deficiencies of genes associated with the ANT-C have profound effects on embryogenesis. The mutant phenotypes suggest, in addition to ensuring proper segment identity, the wild-type alleles of the 84A-84B1,2 genes are necessary for normal segmentation and elongation of the germ band and normal head involution.     

Liver microsomal polypeptides from Fischer-344 rats affected by age,sex, and xenobiotic induction

In order to investigate age and sex as determinants of hepatic cytochromes P-450, the polypeptide compositions of liver smooth microsomes from Fischer-344 rats were examined using two-dimensional gel electrophoresis (G. P. Vlasuk and F. G. Walz, Jr. (1980)Anal. Biochem. 105, 112). The effects of phenobarbital and 3-methylcholanthrene treatments were investigated using sexually immature (1 month), young adult (3 months), middle aged (12 months), and senescent (26 months) animals of both sexes. The appearance of five major microsomal polypeptides characterized sexual maturation in males. The only qualitative difference in the patterns of xenobiotic-induced polypeptides were found for young adult and middle-aged males where cytochrome P-450a (D. Ryan, P. E. Thomas, D. Korzeniowski, and W. Levin (1979)J. Biol. Chem. 254, 1365) was not induced by phenobarbital. A number of major microsomal polypeptides which might represent unidentified forms of cytochrome P-450 in untreated males and females were markedly decreased in a specific manner as a result of phenobarbital and/or 3-methylcholanthrene treatments. Microsomes from females of all ages tested and immature males were essentially indistinguishable on the basis of their total cytochrome P-450 contents and polypeptide patterns. Untreated senescent males were characterized by a reversion of their microsomal polypeptide patterns and total cytochrome P-450 contents to those for females and sexually immature males. In addition, phenobarbital-induced levels of total cytochrome P-450 for senescent males were the lowest observed for all of the groups tested even though their pattern of induced polypeptides was qualitatively the same as that for females.     

A specific inhibitor to δ-aminolevulinate dehydratase in rat bone marrow cells

A factor that specifically inhibited δ-aminolevulinate dehydratase was found in rat bone marrow cells. The inhibitor, which was located in the supernatant fraction of the bone marrow hemolysate, was purified about 12-fold by ammonium sulfate fractionation and column chromatography on Sephadex G-75. The partially purified inhibitor was heat labile and sensitive to trypsin and was denatured by urea. It had a pH optimum of 7.5–8.0, and a molecular weight of 28,000. It inhibited the activity of δ-aminolevulinate dehydratase noncompetitively.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.     

Purification and characterization of chitinase from the stable fly,Stomoxys calcitrans

Chitinase that appears as a single band by electrophoresis was purified from stable fly pupae. The chitinase has no cation requirements for activity, and a broad pH optimum around 5. The molecular weight of the chitinase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 48,000, and the isoelectric point is 4.85. Kinetic properties were determined using acetylated chitosan. The K_m is 33 mm and V is 1.21 μmol/min/mg protein. The insect growth regulator diflubenzuron had no effect on chitinase activity.     

Purification and characterization of intact cytochrome b5 from yeast microsomes

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.     

Endo-(1→4)-β-d-glucanases from sclerotium rolfsii. Purification,substrate specificity,and mode of action

Three purified endo-(1→4)-β-d-glucanases (EC 3.2.1.4), A, B, and C, from Sclerotium rolfsii culture filtrates showed homogeneity in disc-gel electrophoresis and in analytical isoelectric-focusing in polyacrylamide gel. The three endo-d-glucanases are glycoproteins, endo B and endo C being composed of a single polypeptide chain, and endo A of two dissimilar polypeptide chains that are covalently bound by a disulfide bridge. Endo B and endo C do not contain half-cystine residues. With carboxymethylcellulose as substrate, the liquifying activity of the three enzymes was inhibited by cellobiose but not by d-glucose. The specificity of the enzymes is restricted to β-(1→4) linkages, but they showed some differences in the mode of attack on cellodextrins, phosphoric acid-swollen cellulose, and lichenan to give cellobiose, cellotriose, and small proportions of d-glucose. Endo B in addition showed endo-d-xylanase activity.     

Modification of the reactive sulfhydryl of bacterial luciferase with spin-labeled maleimides

Concerned about the lack of agreement among reports of the molecular weight of ferredoxin-NADP⁺ oxidoreductase, we carried out molecular weight determinations in the ultracentrifuge. Sedimentation velocity, sedimentation equilibrium, and active enzyme sedimentation studies all indicate that ferredoxin-NADP⁺ oxidoreductase in the purified form is active as a monomer of molecular weight 33,000–36,000.