bluuebunny/crossref_metadata_2025 huggingface dataset:
The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans
Authors: Alefounder P R , Ferguson S J
Published: August 2015

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.
Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid
Authors: Schmidt E , Knackmuss H J
Published: August 2015

1. An enzyme for the cycloisomerization of 2- and 3-chloro-cis, cis-muconic acid was isolated from 3-chlorobenzoate-grown cells of Pseudomonas sp. B13. It was named muconate cycloisomerase II, because it could it clearly be differentiated by its Km and Vmax. values from an ordinary muconate cycloisomerase, which functioned in benzoate catabolism and exhibited low activity with the chlorinated substrates. 2-Chloro-cis, cis-muconic acid was converted into trans- and 3-chloro-cis, cis–muconic acid into cis-4-carboxymethylenebut-2-en-4-olide together with dehalogenation. 2. An enzyme was isolated from chlorobenzoate-grown cells, which converted the 4-carboxymethylenebut-2-en-4-olides into maleoylacetic acid.
Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion, endocytosis or fusion?
Authors: Hallett Maurice B. , Campbell Anthony K.
Published: August 2015

1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome–cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca2+ ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca2+ seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500μm). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca2+ was developed in which an anti-cell anti-body–complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5′-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca2+ occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5′-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.
The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine
Authors: Schlesinger P H , Rodman J S , Doebber T W , Stahl P D , Lee Y C , Stowell C P , Kuhlenschmidt T B
Published: August 2015

The mannose- and N-acetylglucosamine-specific pathway for the clearance of mammalian glycoproteins has been characterized by using 125I-labelled neoglycoproteins, glycosidase-treated orosomucoid and lysosomal glycosidases (beta-glucuronidase and beta-N-acetylglucosaminidase) as probes. There are two components to this pathway in vivo; one liver-dependent and the other extrahepatic or liver-independent. Cells that mediate clearance by the latter component of the pathway are present in spleen, bone and in elements of the reticuloendothelial system, but not in the kidney. Glycoproteins that possess terminal mannose, glucose or N-acetylglucosamine residues, including various lysosomal enzymes, are rapidly cleared from plasma via this pathway. Glucose-terminated glycoproteins are recognized by two pathways in the intact animal; the hepatic galactose-specific pathway and the mannose/N-acetylglycosamine-specific pathway, which is present in liver and in peripheral tissues. Following removal of the liver by surgical evisceration, glucose-terminated glycoproteins are cleared whereas glycoproteins bearing galactose are not cleared. Uptake of 125I-labelled neoglycoproteins and agalacto-orosomucoid by isolated alveolar macrophages closely mimics clearance in vivo by the mannose/N-acetylglucosamine pathway. Neoglycoproteins terminated by mannose, glucose or N-acetylglucosamine all compete with 125I-labelled agalacto-orosomucoid for uptake by receptor-mediated pinocytosis. The extent of substitution of the neoglycoproteins is a critical determinant of their inhibitory potency. It is proposed that mononuclear phagocytes are in important component of the clearance pathway in vivo. The mannose/N-acetylglucosamine pathway may be important in the regulation of extracellular levels of various glycosylated macromolecules, including lysosomal hydrolases.
The metabolism of l-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism
Authors: Smith S A , Carr F P A , Pogson C I
Published: August 2015

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5–6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.
The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis
Authors: Tyler David D.
Published: August 2015

1. The distribution of Pi between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J.111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of Pi transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total Pi liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the Pi liberated was retained in the mitochondria. 2. Larger amounts of Pi appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this Pi were identified: (a) a slow efflux of Pi from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg2+, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and Pi and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and Pi, is unaffected by uncoupling agents and is activated by endogenous or added Mg2+. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the Pi liberated was recovered in the suspending medium, unless inhibitors of Pi-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg2+ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of Pi retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the Pi transporter provides the sole pathway for Pi efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg2+ on mersalyl inhibition of the Pi transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in Pi efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A.75, 4788–4792] are erroneous and should be discarded.
Involvement of superoxide anion in the reaction mechanism of haemoglobin oxidation by nitrite
Authors: Tomoda A , Tsuji A , Yoneyama Y
Published: August 2015

The sigmoidal time course of haemoglobin oxidation by nitrite, involving an initial slow reaction accompanied by a subsequent rapid reaction, was extensively explored. The initial slow reaction was much prolonged by the addition of superoxide dismutase to the reaction mixture. On the other hand, in the presence of superoxide anion generated by xanthine oxidase systems, the slow phase disappeared and the reaction changed to first-order kinetics. The oxidation of intermediate haemoglobins [defined as haemoglobin tetramer in which different chains (alpha- or beta-) are in the ferric state and in the ferrous state] such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 also proceeded in a sigmoidal manner. Similar effects of superoxide anion on these reactions were observed. Since the intermediate haemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were found to be produced by the oxidation of haemoglobin by nitrite, the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were followed by isoelectric-focusing electrophoresis. The amounts of (alpha 2+ beta 3+)2 were larger than those of (alpha 3+ beta 2+)2 at the initial stages of the reaction, suggesting that there is a functional difference between alpha- and beta-chains in the oxyhaemoglobin tetramer. On the basis of these results, a reaction model of the haemoglobin oxidation by nitrite was tentatively proposed. The changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin were well fitted to the simulation curves generated from the reaction model. Details of the derivation of the equations used for kinetic analysis have been deposited as Supplement SUP 50112 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K. from whom copies may be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
Studies on the hydrophobic properties of sphingomyelinase
Authors: Callahan J W , Gerrie J , Jones C S , Shankaran P
Published: August 2015

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.
Characterization of collagenous and non-collagenous peptides of a glycoprotein isolated from alveoli of patients with alveolar proteinosis
Authors: Bhattacharyya S N
Published: August 2015

A glycoprotein of Mr 62 000 was isolated from lung lavage material of patients with alveolar proteinosis. The glycoprotein was found to contain (per molecule) 72 residues of glycine, 5 residues of hydroxyproline, 3 molecules of sialic acid, 4.9 molecules of mannose, 4.0 molecules of galactose, 0.9 molecule of fucose and 7.0 molecules of N-acetylglucosamine. Limited pepsin digestion of the glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine, and two of which contained all the carbohydrate present in the glycoprotein of Mr 62 000. The three peptides containing hydroxyproline and with high content of glycine contained a repeating -Gly-X-Y-sequence in the peptide chain. Partial amino acid-sequence analyses on the peptides derived from the digestion of the alveolar glycoprotein with various proteolytic enzymes indicate that this glycoprotein is characterized by the presence of alternating collagenous and non-collagenous regions in the same polypeptide chain.
Fractionation of cell-wall preparations from grass leaves by centrifuging in non-aqueous density gradients
Authors: Gordon A H , Bacon J S
Published: August 2015

1. Dried preparations of cell walls from perennial-ryegrass (Lolium perenne) and Italian-ryegrass (L. multiflorum) leaves were suspended in mixtures of carbon tetrachloride with light-petroleum (b.p. 45–50 degrees C) or alcohols and layered on density gradients formed from the same solvents. 2. On centrifugation, the cell walls become distributed throughout a suitably chosen gradient. Fractions corresponding to various regions of the gradient were separated, examined under the microscope and analysed. 3. Cell-wall preparations made from leaf material ground in liquid N2, or in a triple roll mill, showed considerable heterogeneity in particle size, and their behaviour in the density gradient was variable, although there was a general indication that walls derived from vascular bundles were less dense than those from sclerenchyma. 4 Treatment in a vibratory ball mill decreased the size of the particles and produced a more uniform material, but made it impossible to distinguish the origins of the particles. This material behaved more reproducibly in the density gradient. 5. Some fractionations were also made by successive centrifugation in media of increasing relative density. 6. Analyses of the fractions obtained by each method indicated that the less dense had a greater proportion of xylose in the polysaccharide components, and higher contents of acetyl groups and lignin, confirming the close relationship between these components in plant cell walls. 7. The results show that there are differences in polysaccharide composition between the cell-wall types in the grass leaf, the vascular tissue being richer in hemicellulose relative to cellulose than the sclerenchyma.
Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2
Authors: Srivastava S. , Muhlrad A. , Wikman-Coffelt J.
Published: August 2015

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca2+-binding properties of the light chains. The Ca2+-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH2 thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca2+-binding properties of light chains and generation of tension. When the SH2 moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca2+-binding properties of light chain LC2 is lost; under these conditions the Ca2+-binding affinity value of SH2-N-ethylmaleimide-blocked myosin (3.3×104m−1) decreases to near that expressed with the dissociated light chain LC2 (0.7×104m−1). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH2 thiol does not affect Ca2+ binding. The native secondary and tertiary structure of myosin seem to be required for Ca2+ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH2-N-ethylmaleimide-blocked myosin normal Ca2+- and (Mg2++actin)-stimulated ATPase activities are expressed; however, there is a loss in K+-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca2+ with alterations in pH values. In the absence of Ca2+/EGTA buffer the biphasic Ca2+-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×106m−1 and site two: 0.4×106m−1) as compared with values obtained at pH6.5 (site one: 0.64×106m−1 and site two: 0.2×106m−1). The Ca2+-binding affinity of light chain LC2 and S1, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca2+-binding affinity, approx. 0.7×104m−1, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca2+-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S1 myosin subfragment light chain LC2 was lost and thus was added back to the purified S1 fraction. Light chain LC2 was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC2 and thus influences its essential conformation for Ca2+ binding.
The relationship between the activity of DNA-dependent RNA polymerase I and the rate of synthesis of rRNA in hepatoma cells in culture
Authors: Thompson E A , Keith R H , Cavanaugh A H , Wood K M
Published: August 2015

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate–RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.
Corticotropin-(1–24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain
Authors: Jolles J , Zwiers H , Dekker A , Wirtz K W A , Gispen W H
Published: August 2015

1. Effects of corticotropin-(1–24)-tetracosapeptide on the endogenous phosphorylation of proteins and lipids were studied in a membrane/cytosol fraction prepared from a lysed crude mitochondrial/synaptosomal fraction. 2. The labelling of proteins and lipids was monitored by incubation of the subcellular fraction for 10s with [gamma-32P]ATP. 3. The phosphorylation of proteins was dose-dependently inhibited by the peptide (40% of control incubations at 100 microM-corticotropin). 4. Of the membrane phospholipids only phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidic acid became labelled. Corticotropin dose-dependently increased the formation of phosphatidylinositol bisphosphate and inhibited the production of phosphatidic acid (470% and 50% respectively of control incubations, at 100 microM of the peptide) and had no effect on phosphatidylinositol phosphate. 5. Phosphatase activity was observed to act on phosphatidylinositol bisphosphate, phosphatidylinositol phosphate and phosphoprotein but not on phosphatidic acid. 6. Corticotropin interacted with the kinases rather than with the phosphatases. 7. The formation of phosphatidylinositol bisphosphate and phosphatidic acid was maximal at 1–10mM-Mg2+ in the absence of Ca2+, and the production of phosphatidylinositol phosphate was maximal at 30mM-Mg2+. 8. The basal value of lipid phosphorylation decreased with increasing Ca2+ concentration. 9. Ca2+ abolished the effect of corticotropin on phosphatidylinositol bisphosphate formation (470%, 190% and 100% of control incubations at respectively 0, 0.1 and 1 mM-Ca2+). 10. The data provide evidence that the effects of corticotropin on protein phosphorylation and on polyphosphoinositide metabolism in brain membranes are related.
The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle
Authors: Thrower S , Neethling C , White J O , Lim L
Published: August 2015

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.
Determination of some biochemical and structural features of alcohol dehydrogenases from Drosophila simulans and Drosophila virilis. Comparison of their properties with the Drosophila melanogaster Adhs enzyme
Authors: Juan E , González-Duarte R
Published: August 2015

The biochemical properties of the enzyme alcohol dehydrogenase of two different Drosophila species, Drosophila simulans and Drosophila virilis, were studied and compared with those of Drosophila melanogaster Adhs enzyme. All of them consist of two identical subunits of molecular weight 27800 and share significant similarities in function. The substrate specificities of these enzymes were characterized and Km(app.) and Vmax.(app.) values were calculated. All these alcohol dehydrogenases show greater affinity for secondary rather than for primary alcohols. The amino acid compositions of the three enzymes were determined, and there is a close similarity between the D. simulans and the D. melanogaster enzymes, but there are significant differences from the alcohol dehydrogenase of D. virilis. The N-terminal amino acid is blocked and the C-terminal amino acid is the same for all three alcohol dehydrogenases. The enzymes from the three species were carboxymethylated and digested with trypsin. The peptide ‘maps’ reveal, as expected, more homologies between the enzymes of D. simulans and D. melanogaster than with the enzyme of D. virilis.
Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons
Authors: McKay G , Shargool P D
Published: August 2015

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.
High-molecular-weight catecholamine–ATP aggregates are absent from the chromaffin-granule aqueous phase
Authors: Sen R , Sharp R R
Published: August 2015

Direct n.m.r. studies show that the viscous liquid phase which separates from solutions containing physiological concentrations of noradrenaline, ATP and Ca2+ is not present in the chromaffin-granule interior. This finding is verified for both adrenaline and noradrenaline by using 13C n.m.r. spectra.
Dianthins, ribosome-damaging proteins with anti-viral properties from Dianthus caryophyllus L. (carnation)
Authors: Stirpe F , Williams D G , Onyon L J , Legg R F , Stevens W A
Published: August 2015

1. Dianthin 30 and dianthin 32, two proteins isolated from the leaves of Diathus caryophyllus (carnation), were purified to homogeneity by chromatography on CM-cellulose. 2. The mol.wt. of dianthin 30 is 29 500 and that of dianthin 32 is 31 700. Both dianthins are glycoproteins containing mannose. 3. Dianthins inhibit protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 9.15 ng/ml (dianthin 30) and 3.6 ng/ml (dianthin 32). They act by damaging ribosomes in a less-than-equimolar ratio. Protein synthesis by intact cells is partially inhibited by dianthins at a concentration of 100 microgram/ml. 4. Dianthins mixed with tobacco-mosaic virus strongly decrease the number of local lesions on leaves of Nicotiana glutinosa.
The role of the arginine-B22 residue in insulin action
Authors: Rose K , Rees A R , Drake C S , Offord R E
Published: August 2015

We describe the modification of the side chain of the arginine-B22 residue of insulin by the N8N9-(1, 2-dihydroxycyclohex-1,2-ylene) group and by the adipoyl group. These are the first insulin derivatives described that contain a modified arginine residue in an otherwise unaltered molecule. When tested for their ability to lower blood sugar concentration, both modified insulins showed a specific activity indistinguishable from that of insulin. In view of the fact that the substituent groups involved are very bulky and in one case of opposite charge to that of the side chain, the retention of biological activity casts doubt on the idea, previously generally accepted, that the arginine-B22 residue is essential to the activity of the hormone.
Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase
Authors: Maines Mahin D.
Published: August 2015

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of γ-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.
Studies on the efflux of metalloporphyrin from rat liver mitochondria. Effect of K+ and other cations
Authors: Husby P , Romslo I
Published: August 2015

The mechanism by which metalloporphyrins synthesized within the mitochondria escape to the incubation medium was studied in isolated rat liver mitochondria. In a low-ionic-strength sucrose medium, the efflux of metalloporphyrins is markedly decreased when K+ (greater than 10 mM) is added. The effect of K+ is not dependent on the energy state of the mitochondria and it can in part be abolished by adding globin, but not albumin. K+ also decreases the uptake of porphyrins by the mitochondria and thereby the rate of synthesis of metalloporphyrins. Qualitatively similar results are found with Na+, Li+, Mg2+ and Ca2+. Quantitatively, however, the efficiency of cations to inhibit the release of metalloporphyrins decreases in the order: Mg2+ greater than Ca2+ greater than K+ greater than Li+ greater than Na+. Co-protoporhyrin behaves essentially as Co-deuteroporphyrin. The results provide further evidence that the efflux of metalloporphyrins from the mitochondria depends on haem-binding ligands of the suspending medium and also on the ionic strength of the incubation medium.
Differential labelling of UDP-N-acetylglucosamine in Huntington's-chorea fibroblasts
Authors: Hung W Y , Tourian A
Published: August 2015

The hypothesis that there is impaired endogenous synthesis of glucosamine 6-phosphate in Huntington's-chorea fibroblasts was tested by double labelling matched pairs of fibroblasts in culture with carrier-free H3 32PO4 and [U-14C]glucosamine. The [32P]UDP-N-acetyl[14C]glucosamine and [14C]glucosamine 6-[32P]phosphate of the cellular soluble fraction was isolated by charcoal column and paper chromatography. There is no quantitative difference in 32P but a significant difference in 14C in these two sugars in a ratio of approx. 1.5 for Huntington's-chorea fibroblasts compared with normal fibroblasts.
Labelling by axonal transport of myelin-associated proteins in the rabbit visual pathway
Authors: Giorgi P P , DuBois H
Published: August 2015

After intraocular injections of [3H]leucine, six regions of the visual pathway of adult rabbit were used to study the spatio-temporal pattern of the slow anterograde axonal transport of radioactive proteins associated with the particulate fraction, the water-soluble fraction and the myelin fraction. Unlike other fractions, myelin-associated labelled proteins represented a time-constant (for a given region) percentage of total tissue radioactivity. This percentage increased from the first half to the second half of the optic nerve and remained high in the chiasma and tract. The peak specific radioactivity of myelin decreased in the same direction. Myelin proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the labelling patterns obtained in different regions and at different survival times were compared. At the peak of myelin radioactivity of a given region the label was typically associated with four protein bands, L1, L2, L3 and L4, of 40000, 44000, 62000, and 68000 mol.wts. respectively. The basic protein, the proteolipid protein and the W1 component (mol.wt. 51000-53000) of the Wolfgram proteins were not significantly labelled. The radioactivity associated with the W2 component (mol.wt 60000) of the Wolfgram proteins could be derived from the closely migrating L3 component. At shorter survival times no clear labelling pattern could be detected. At longer survival times radioactivity was almost totally localized around band L3. The results presented underline the importance of choosing appropriate experimental conditions to obtain a consistent labelling pattern of myelin-associated proteins and to investigate the possible mechanism responsible for this phenomenon.
An alternative pathway for the biosynthesis of isoprenoid compounds in bacteria
Authors: Pandian S , Saengchjan S , Raman T S
Published: August 2015

The pattern of incorporation of radioactivity from [1-14C]acetate and [2-14C]acetate into the polyprenyl side-chain of ubiquinones in bacteria (Azotobacter vinelandii, Pseudomonas sesami, Escherichia coli and Rhodopseudomonas capsulata) was studied. For this purpose, a new degradation method involving a modified Barbier-Wieland reaction of laevulinic acid was developed, and used along with the iodoform reaction. Both C-1 and C-2 of acetate were incorporated exclusively into C-2 of laevulinic acid suggesting that the well-known pathway through acetoacetyl-CoA (‘acetoacetate pathway)’ was not operative in these bacteria. An alternative pathway (‘acetolactate pathway’), starting with pyruvate and acetaldehyde as the distal precursors, and utilizing the reactions of leucine and valine metabolism, was postulated. It was also postulated that C-1 of acetate is incorporated not directly, but after oxidation to CO2. The pattern of incorporation of radioactivity from [U-14C]valine, [U-14C]alanine and NaH14CO3 into the side-chain of ubiquinone of R. capsulata was in agreement with the operation of the ‘acetolactate pathway’.
Heparin-releasable lipase activity of rat adrenals, ovaries and testes
Authors: Jansen H , De Greef W J
Published: August 2015

The presence of NaCl-resistant, neutral triacylglycerol hydrolase (lipase) activity in rat adrenal gland, ovary and testis was studied. Both adrenals and ovaries but not testes were found to contain such a lipase. The activity of the enzyme in the adrenal gland was lowered during cortisol treatment and hypothyroidism. An elevated adrenal lipase activity was found during hyperthyroidism. Pseudo-pregnant and lactating rats had higher ovarian lipase activities than cyclic rats. Ovarian lipase activity in lactating rats was positively correlated with the serum concentrations of progesterone and of 20 alpha-hydroxyprogesterone and negatively correlated with the high-density-lipoprotein non-esterified cholesterol concentration. The lipase activity of adrenals and of ovaries was largely releasable from these organs by heparin and could be inhibited by an antibody against heparin-releasable liver lipase. This indicated that the lipase is extracellularly located and is similar to ‘liver’ lipase. A possible role of this lipase in adrenals and ovaries is discussed.
Effects of trifluoperazine and pimozide on stimulus–secretion coupling in pancreatic B-cells Suggestion for a role of calmodulin?
Authors: Henquin Jean-Claude
Published: August 2015

The possible involvement of calmodulin in insulin release was evaluated by studying the effects on intact islets of trifluoperazine and pimozide, two antipsychotic agents known to bind strongly to calmodulin in cell-free systems. Trifluoperazine (10–100μm) produced a dose- and time-dependent inhibition of the two phases of glucose-stimulated insulin release. The effect was not reversible by simple washing of the drug, but could be prevented by cytochalasin B or theophylline. Trifluoperazine also inhibited the release induced by glyceraldehyde, oxoisocaproate, tolbutamide or barium, but not that stimulated by 10mm-theophylline or 1mm-3-isobutyl-1-methylxanthine. Pimozide (0.5–10μm) also produced a dose-dependent inhibition of insulin release triggered by glucose, leucine or barium, but did not affect the release induced by methylxanthines. Glucose utilization by islet cells was not modified by trifluoperazine (25μm), which slightly increased cyclic AMP concentration in islets incubated without glucose. The drug did not prevent the increase in cyclic AMP concentration observed after 10min of glucose stimulation, but suppressed it after 60min. Basal or glucose-stimulated Ca2+ influx (5min) was unaffected by 25μm-trifluoperazine, whereas Ca2+net uptake (60min) was inhibited by 20%. Glucose-stimulated Ca2+ uptake was almost unaffected by pimozide. In a Ca2+-free medium, trifluoperazine decreased Ca2+ efflux from the islets and did not prevent the further decrease by glucose; in the presence of Ca2+, the drug again decreased Ca2+ efflux and inhibited the stimulation normally produced by glucose. In the absence of glucose, trifluoperazine lowered the rate of Rb+ efflux from the islets, decreased Rb+ influx (10min), but did not affect Rb+ net uptake (60min). It did not interfere with the ability of glucose to decrease Rb+ efflux rate further and to increase Rb+ net uptake. The results show thus that trifluoperazine does not alter the initial key events of the stimulus–secretion coupling. Its inhibition of insulin release suggests a role of calmodulin at late stages of the secretory process.
Effect of a transitory ischaemia on the structure-linked latency of rat liver acid phosphatase and β-galactosidase
Authors: Wattiaux R , Wattianx-De Coninck S
Published: August 2015

The structure-linked latency of acid phosphatase and beta-galactosidase was studied in rat liver lobes made ischaemic for 1 or 2 h and then recirculated with blood for increasing periods. Free activity of acid phosphatase and unsedimentable activity of beta-galactosidase are increased in homogenates of ischaemic livers. When ischaemia had been maintained for 1 h, the recovery of normal latency for both enzymes was observed 1 h after re-establishment of the blood flow. After a 2 h period of ischaemia, unmasked activity markedly decreases during the first 1 h after restoration of blood flow; after that, a large and irreversible secondary rise takes place. Chlorpromazine, injected 30 min before or just after induction of ischaemia, extensively prevents the latency decrease occurring during restoration of blood flow. Modifications of the hydrolase distribution pattern obtained after differential centrifugation are in agreement with the latency changes. These results suggest that a 2 h ischaemia causes an alteration of the liver lysosomes that is largely reversible and that restoration of blood flow induces an irreversible alteration of these organelles. Chlorpromazine treatment prevents the irreversible lesion from taking place.
Regulation of hepatic fatty acid metabolism The activities of mitochondrial and microsomal acyl-CoAsn-glycerol 3-phosphate O-acyltransferase and the concentrations of malonyl-CoA, non-esterified and esterified carnitine, glycerol 3-phosphate, ketone bodies and long-chain acyl-CoA esters in livers of fed or starved pregnant, lactating and weaned rats
Authors: Zammit Victor A.
Published: August 2015

1. The concentrations of malonyl-CoA, glycerol 3-phosphate, non-esterified carnitine, acid-soluble and acid-insoluble acylcarnitines, acetoacetate, 3-hydroxybutyrate and acid-insoluble acyl-CoA were measured in rapidly-frozen liver samples from fed or starved (24h) virgin, pregnant (19–20 days), lactating (2, 10–12 and 18–20 days) and weaned (for 24h, on 10th day of lactation) rats. The activities of total and N-ethylmaleimide-sensitive and -insensitive glycerophosphate acyltransferase (acyl-CoA:sn-glycerol 3-phosphate O-acyltransferase; EC 2.3.1.15) were also measured. 2. The concentration of malonyl-CoA was significantly higher in liver of fed pregnant, mid- and late-lactating rats than in liver of fed virgin rats. After starvation for 24h hepatic malonyl-CoA concentrations were higher in mid-lactating rats and lower in pregnant and weaned rats than in virgin animals. 3. After starvation for 24h the hepatic concentrations of glycerol 3-phosphate, ketone bodies, acid-soluble acylcarnitines and the value for the [3-hydroxybutyrate]/[acetoacetate] ratio were all highest in pregnant rats, intermediate in virgin, 2-day lactating and weaned animals and lowest in mid- and late-lactating rats. The concentrations of acid-insoluble acylcarnitines also increased most in pregnant rats, after starvation. The concentration of acid-insoluble acyl-CoA increased equally after starvation in virgin and pregnant animals but did not increase significantly in all other animals studied. 4. The total concentration of carnitine was similar in livers of fed virgin, pregnant and 2-day lactating animals but fell markedly by the 10th day of lactation and remained low in late-lactating animals. The concentration of non-esterified carnitine followed the same pattern. After starvation for 24h the hepatic concentration of non-esterified carnitine decreased significantly in virgin, pregnant and 2-day lactating animals, but remained unchanged in mid- and late-lactating or weaned animals. 5. The activities of N-ethylmaleimide-sensitive and -insensitive glycerophosphate acyltransferase both increased significantly in livers of mid-lactating animals. After starvation for 24h the activity of the N-ethylmaleimide-insensitive O-acyltransferase decreased in livers of virgin, pregnant and mid-lactating animals, whereas the activity of the N-ethylmaleimide-sensitive O-acyltransferase was unchanged in virgin animals but decreased markedly in livers of pregnant and lactating rats. 6. The results are discussed in relation to the importance of different metabolic parameters in the regulation of long-chain acyl-CoA metabolism in the liver.