bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormonal stimulation of cyclic AMP accumulation and glycogen phosphorylase activity in calcium-depleted hepatocytes from euthyroid and hypothyroid rats
Authors: Malbon C C , Gilman H R , Fain J N
Published: August 2015

Activation of glycogen phosphorylase by hormones was examined in hepatocytes isolated from euthyroid and hypothyroid female rats and incubated by Ca2+-free buffer containing 1 mM-EGTA. Basal glycogen phosphorylase activity was decreased in Ca2+-free buffer. However, the activation of hepatocyte glycogen phosphorylase, in the absence of extracellular Ca2+, in response to adrenaline, glucagon or phenylephrine was slightly lower, whereas that by vasopressin was abolished. The activation of glycogen phosphorylase by phenylephrine, adrenaline or isoproterenol (isoprenaline) in hepatocytes from euthyroid rats incubated in the absence of Ca2+ was not accompanied by any detectable increase in total cyclic AMP. The log-dose/response curves for activation of phosphorylase by phenylephrine or low concentrations of adrenaline were the same in hepatocytes from hypothyroid as compared wit euthyroid rats, whereas the response to isoproterenol was greater in hepatocytes from hypothyroid rats. However, the increases in total cyclic AMP accumulation caused by adrenaline or isoproterenol were greater in hepatocytes from hypothyroid rats than in hepatocytes from euthyroid rats. The increases in cyclic AMP accumulation caused by adrenaline or isoproterenol in Ca2+-depleted hepatocytes from hypothyroid rats were blocked by propranolol, a beta-adrenergic antagonist. In contrast, propranolol was only partially effective asan inhibitor of the activation of glycogen phosphorylase by phenylephrine or adrenaline in hepatocytes from hypothyroid rats and ineffective on phosphorylase activation in cells from euthyroid rats. These data indicate that the alpha-adrenergic activation of glycogen phosphorylase is not affected by the absence of extracellular Ca2+, and the extent to which total cyclic AMP was increased by adrenergic amines did not correlate with glycogen phosphorylase activation.
Changes in polyribosomes and poly(A)-containing polyribosomal RNA in the uterus of the ovariectomized rat in response to oestradiol-17 β treatment
Authors: Pretorius P J , Reinecke C J
Published: August 2015

Polyribosome formation and the characteristics of polyribosomal poly(A)-containing RNA from uteri of ovariectomized rats responding to a single dose of oestradiol-17 beta was investigated. The mean proportion of polyribosomes in the atrophic uterus was 65%. In response to 10 micrograms of oestradiol-17 beta/100 g body mass, the amount of polyribosomes increased to 88% 24 h after stimulation. Thereafter the proportion of polyribosomes decreased to a value of 48% at 72h. The pattern of amino acid incorporation in oocytes from Xenopus laevis injected with these polyribosomes was similar to the changes in polyribosome formation and degradation. The polyribosomal poly(A)-containing RNA from the controls consisted of a heterogeneous population of RNA with sedimentation values between 5S and 25S. The hormone stimulation resulted in an increase in both the amount and the size (13S to 35S) of the RNA.
Regeneration of cilia in starved Tetrahymena thermophila involves induced synthesis of ciliary proteins but not synthesis of membrane lipids
Authors: Skriver L , Williams N E
Published: August 2015

The synthesis of ciliary-membrane phospholipids and ciliary proteins was studied after deciliation in starving Tetrahymena thermophila cells. Deciliated cells regenerated the new ciliary membrane without any induced phospholipid synthesis. The constant cell volume found during the regrowth of the cilia suggests that renewal of ciliary membranes takes place by insertion of intracellular membrane material into the cell surface. In contrast with the absence of induced phospholipid synthesis during ciliary regeneration, the synthesis of ciliary proteins was found to be induced. This enhanced synthetic activity was made possible by an increased rate of intracellular protein degradation in regenerating cells. It was found that the extent of the induced synthesis strongly depends upon the growth conditions of the cells before starvation. Furthermore, it was shown that the degree of induced protein synthesis is greater for higher-molecular-weight ciliary proteins than for lower-molecular-weight species.
Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver
Authors: Reddy M. Kumudavalli , Hollenberg Paul F. , Reddy Janardan K.
Published: August 2015

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun.89, 580–584].
Secretion granules of transplantable pancreatic acinar carcinoma of rat
Authors: Reddy J K , Reddy M K , Hansen L J , Qureshi S A
Published: August 2015

Secretion granules of the rat transplantable pancreatic acinar carcinoma and of normal rat pancreas were isolated by differential centrifugation. Analysis of the granule content by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing procedures combined with specific enzyme assays indicated essential qualitative similarities between normal and neoplastic secretory proteins, suggesting retention of enzymic differentiation in this pancreatic acinar carcinoma. Accordingly, this epithelial tumor should serve as an important model for examination of regulatory mechanisms in cell differentiation and neoplasia.
Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides
Authors: Henderson C E , Perham R N
Published: August 2015

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.
Polypeptide heterogeneity of hamster and calf fibronectins
Authors: Pena S D J , Mills G , Hughes R C , Aplin J D
Published: August 2015

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide ‘maps’. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].
Configuration and interactions of the polar head group in gangliosides
Authors: Maggio Bruno , Cumar Federico A. , Caputto Ranwel
Published: August 2015

1. The interactions of gangliosides with Ca2+ and some polar-head-group requirements for establishment of particular interactions with phosphatidylcholine were studied in monolayers at the air/145mm-NaCl interface. 2. Ganglioside–Ca2+ interactions, as revealed by surface-potential measurements, depended on the position occupied by sialosyl residues in the oligosaccharide chain. The interactions with Ca2+ of the single sialosyl residue of monosialogangliosides occurred above 0.1mm-CaCl2, whereas the interaction of the cation with additional sialosyl groups in di- or tri-sialogangliosides depended on the carbohydrate residue to which the sialosyl moiety was attached. The sialosyl residue bound in sialosyl–sialosyl linkage interacted very little with Ca2+. The sialosyl residue attached to the terminal galactose of the neutral tetrasaccharide chain interacted with Ca2+ above 1μm-CaCl2. 3. Experiments with mixed monolayers containing dihexadecyl phosphate and hexadecyltrimethylammonium indicated that for the occurrence of interactions of polysialogangliosides with phosphatidylcholine characterized by reductions in molecular packing and surface potential both charged groups of the phospholipid and sialosyl residues with particular dipolar properties in the ganglioside are participating. 4. Possible configurations that can explain the behaviour in monolayers were inspected with space-filling molecular models. The position of the carboxylate group of sialosyl residues with respect to the interface and to the sialosyl molecular plane can explain the different orientation of the dipole-moment vector of this residue, which depends on the position to which it is linked in the oligosaccharide chain. Favoured interactions of polysialogangliosides with phosphatidylcholine may result from a configuration allowing a partial matching of two oppositely oriented electrical vectors contributed by the zwitterionic phosphocholine group and particular sialosyl groups.
Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms
Authors: Danielsen E M , Norén O , Sjöström H , Ingram J , Kenny A J
Published: August 2015

Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl substrate were attacked most rapidly and their hydrolyses were stimulated by Ca2+. The 2-naphthylamide derivatives of neutral and basic amino acids were also hydrolysed by aminopeptidase A, but at rates about two orders of magnitude lower, and Ca2+ was inhibitory. The possibility that these atypical substrates were hydrolysed by traces of aminopeptidase M (EC 3.4.11.2) contaminating the preparation could be excluded on several grounds. Aminopeptidase A was sensitive to inhibition by chelating agents and the inactive enzyme could be reactivated by Ca2+ or Mn2+. Atomic absorption spectrophotometry revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000–400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar protein.
Different turnover of fucose residues in plasma membranes of rat liver and Morris hepatoma 7777
Authors: Vischer P , Reutter W
Published: August 2015

Half-lives and rate constants of degradation of protein-bound fucose have been determined in plasma membranes and total cell homogenates of rat liver and Morris hepatoma 7777. The existence of at least two dynamically different classes of fucose-containing glycoproteins could be demonstrated in both liver and hepatoma plasma membranes. The apparent half-lives were 8.4 and 24.5 h (host liver) and 11.5 and 33.9 h (Morris hepatoma). Since this biphasic loss of fucose residues was not observed for sialic acid [Harms & Reutter (1974) Cancer Res. 34, 3165–3172], the differences are possibly related to specific functions of fucosylated glycoproteins of the plasma membrane.
Evidence that catecholamines stimulate renal gluconeogenesis through an α1-type of adrenoceptor
Authors: Kessar P , Saggerson E D
Published: August 2015

1. Noradrenaline stimulates gluconeogenesis through an alpha-adrenoceptor in renal cortical tubule fragments from fed rats incubated with 5 mM-lactate. 2. The selective alpha 1-adrenoreceptor agonist methoxamine stimulated gluconeogenesis, but the selective alpha 2-adrenoceptor agonist clonidine was ineffective. 3. The selective alpha 1-adrenoceptor antagonist thymoxamine blocked the stimulatory effects on gluconeogenesis of noradrenaline and of oxymetazoline (a synthetic alpha-agonist). The selective alpha 2-adrenoceptor antagonist yohimbine was ineffective in this respect. 4. It is concluded that noradrenaline and oxymetazoline stimulate gluconeogenesis in rat kidney via an alpha 1-rather than an alpha 2-type of adrenoceptor.
The orientation of iron–sulphur clusters in membrane multilayers prepared from aerobically-grown Escherichia coli K12 and a cytochrome-deficient mutant
Authors: Blum Haywood , Poole Robert K. , Ohnishi Tomoko
Published: August 2015

1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron–sulphur cluster at gx=1.92, gy=1.93 and gz=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the gy axis perpendicular to the membrane plane and with the gx and gz axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron–sulphur protein (Hipip) is seen whose gz=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its gz axis in the membrane plane, whereas a g=2.04 signal is from a cluster with the gz axis lying along the membrane normal. 6. Membrane particles from a glucose-grown, haem-deficient mutant contain dramatically-lowered levels of cytochromes and exhibit, in addition to the iron–sulphur clusters seen in the parental strain, a major signal at g=1.90. 7. Only the latter may be demonstrated to be oriented in multilayer preparations from the mutant. 8. Comparisons are drawn between the orientations of the iron–sulphur proteins in the cytoplasmic membrane of E. coli and those in mitochondrial membranes. The effects of diminished cytochrome content on the properties of the iron–sulphur proteins are discussed.
Factors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration, organelle integrity and ATP
Authors: Thomas J , Debeer L J , De Schepper P J , Mannaerts G P
Published: August 2015

1. The first dehydrogenation step of peroxisomal beta-oxidation involves the reduction of O2 to H2O2. Production rates of H2O2 and acetyl units by purified rat liver peroxisomes oxidizing palmitoyl-CoA were equal, indicating that H2O2 production is a reliable index for the release of acetyl units during peroxisomal fatty-acid oxidation. 2. Measurements of H2O2 and acid-soluble oxidation products during [1-14C]palmitoyl-CoA oxidation by purified peroxisomes revealed that the number of acetyl units released per molecule of palmitoyl-CoA oxidized rapidly decreased with increasing unbound palmitoyl-CoA concentrations. Structural damage to the peroxisomes caused by detergents or other treatments also decreased the number of acetyl units released. Under conditions where oxidation proceeded linearly with time the theoretical maximum of 5 acetyl units released per molecule of palmitoyl-CoA oxidized [Lazarow (1978) J. Biol. Chem. 253, 1522—1528] was never reached. 3. Expressed in terms of acetyl units produced and measured at low unbound-palmitoyl-CoA concentrations, mitochondrial oxidation was 10—20-fold higher than peroxisomal oxidation. 4. ATP stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. The ATP effect required the presence of Mg2+ and was lost when peroxisomal membranes were disrupted by Triton X-100 or high concentrations of unbound palmitoyl-CoA. 5. Disruption of peroxisomes by detergents, freeze—thawing, osmotic or mechanical treatment did not stimulate palmitoyl-CoA oxidation in the presence of ATP, indicating that peroxisomal fatty-acid-CoA oxidation was not latent. In the absence of ATP, Triton X-100 stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold.
NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes
Authors: Petcu L G , Plaut G W E
Published: August 2015

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35—84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine—2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.
A comparison of the relationships between progestin receptors and oestrogen receptors in neural and non-neural target tissues of the rat during the oestrous cycle
Authors: Thrower S , Lim L
Published: August 2015

Similar cyclic changes in the content of nuclear oestrogen receptor occurred in the hypothalamus, cerebral cortex, uterus and pituitary during the oestrous cycle. The relationship of the unoccupied to the total nuclear oestrogen receptor at each phase was similar in all these tissues. However, cyclic changes in the content of the cytosol progestin receptor occurred only in the uterus and pituitary (where they paralleled changes in the nuclear oestrogen receptor), but not in the hypothalamus or cerebral cortex.
Ribosomal resistance to the 12,13-epoxytrichothecene antibiotics in the producing organism Myrothecium verrucaria
Authors: Hobden A N , Cundliffe E
Published: August 2015

An extract of Myrothecium verrucaria, a fungus which produces a range of 12,13-epoxytrichothecene toxins, was found to be resistant to T-2 toxin, one of its products. The epoxytrichothecenes are inhibitors of eukaryotic protein synthesis and normally bind to the 60S ribosomal subunit so as to inhibit peptidyltransferase activity. Ribosomes from M. verrucaria contain 60S subunits which are not subject to inhibition by T-2 toxin and are also resistant to certain other drugs such as anisomycin and homoharringtonine, but not sparsomycin or cycloheximide.
Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali
Authors: Sjöberg Ingrid , Fransson Lars-Ȧke
Published: August 2015

1. 3H- and 35S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. 3H- and 35S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.
The EcoRI restriction endonuclease with bacteriophage λ DNA. Kinetic studies
Authors: Halford S E , Johnson N P , Grinsted J
Published: August 2015

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.
Heterogeneity of proteins resembling high-mobility-group protein HMG-T in trout testes nuclei
Authors: Brown E , Goodwin G H , Mayes E L V , Hastings J R B , Johns E W
Published: August 2015

Two proteins, HMG-T1 and HMG-T2, with electrophoretic mobilities and compositions similar to those of protein HMG-T, were isolated from trout testes nuclei. The isoelectric points of proteins HMG-T1, HMG-T2 and HMG-T differ. The first 20 residues of protein HMG-T2 have been sequenced and differ from protein HMG-T by only one residue.
Albumin associated with purified pig lymphocyte plasma membrane
Authors: Owen M J , Barber B H , Faulkes R A , Crumpton M J
Published: August 2015

Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar “fingerprints” of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.
Variability of mammalian liver nuclear-envelope preparations
Authors: Agutter P S , Gleed C D
Published: August 2015

The composition, density and enzymic activities of sheep liver nuclear-envelope preparations were found to vary markedly according to the concentrations of nuclei during the lysis stage. The effect of nuclear concentration on the properties of the purified envelopes could not be attributed to bound Mg2+ or to other ions, and appeared to result from some component of the nucleus which was not eluted during lysis. The implications of these findings for studies on the nuclear envelope are discussed.
Protein degradation during terminal cytodifferentiation. Studies on mammary gland in organ culture
Authors: Wilde C J , Paskin N , Saxton J , Mayer R J
Published: August 2015

1. In mammary gland explants subjected to experimental manipulation, average rates (during 24 h periods) of degradation of fatty acid synthase, casein and cytosol-fraction proteins were measured by a double-isotope method. Rates of degradation of fatty acid synthase were also computed from measurements of changing enzyme amount and rate of synthesis. 2. During the period of most rapid enzyme accumulation there is a transient decrease in the computed rate of degradation of fatty acid synthase. Removal of hormones produces a rapid increase in the computed rate of degradation of the enzyme. 3. The average rate of degradation of fatty acid synthase measured by the double-isotope method is low in the presence of hormones, and increases on hormone removal. This increase in degradation rate is inhibited by adrenaline and further blocked by insulin. NH4Cl (10 mM) also partially inhibits the increase in protein degradation on hormone removal. 4. The pattern of changes in the average rate of degradation of cytosol-fraction proteins is similar to that for fatty acid synthase alone. There is no relationship between subunit molecular weight and rate of degradation under all experimental conditions. 5. Isotope ratios for resolved cytosol protein mixtures are transformed logarithmically to make the standard deviations an estimate of heterogeneity of degradation rates. By this analysis, in some conditions there appears to be significant measureable heterogeneity of degradation rates. 6. Little degradation of casein is measured in the presence of hormones, but a marked increase in the rate of degradation can be measured when hormones are removed. Whereas at 24-48h NH4Cl (10 mM) has little effect on this enhanced rate of degradation, at 48-72h it causes a large decrease in degradation rate. 7. Results are discussed in terms of a two-component degradation system in mammary gland explants.
Cholesteryl esters are bound by a peptide-initiation and a peptide-elongation factor
Authors: Tuhácková Z , Hradec J
Published: August 2015

Significantly higher quantities of cholesteryl 14-methylhexadecanoate than of cholesteryl laurate and cholesteryl palmitate are bound by a homogeneous peptide-initiation factor and purified peptide-elongation factor 1. Cholesteryl 14-methylhexadecanoate may function as a specific allosteric modifier changing the conformation of protein synthesis factors and thus modulating the activity of their binding sites.
Transport of d-fructose and d-galactose into isolated rat hepatocytes
Authors: Craik J D , Elliott K R
Published: August 2015

Transport of D-fructose and D-galactose across the plasma membrane of isolated rat hepatocytes was followed for the net entry of sugars into sugar-free cells at 20 degrees C. Initial rates of transport showed a Michaelis-Menten dependency on sugar concentration, and transport was inhibited by 3-O-methyl-D-glucose in the external medium.
The purification and properties of the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus
Authors: Cross A R , Anthony C
Published: August 2015

The obligate methylotroph Methylophilus methylotrophus contains three distinct soluble cytochromes c. The major cytochromes, cytochrome cH (about 50% of the total) and cytochrome cL (about 42%), were similar in most respects to the cytochromes cH and cL of the facultative methylotroph Pseudomonas AM1 [O'Keeffe & Anthony (1980) Biochem. J. 192, 411-419]. Cytochrome cH had a high isoelectric point, a midpoint redox potential at pH 7.0 of 373 mV and a low molecular weight (8500). The cytochrome cL had a low isoelectric point, a midpoint potential of 310 mV and a molecular weight of 21,000. The third cytochrome, cytochrome cLM, was clearly distinct from cytochromes cH and cL. Like the cytochrome cL of Pseudomonas AM1, the cytochrome cL of M. methylotrophus had the lowest midpoint potential, it reacted most rapidly with methanol dehydrogenase and it combined to the greatest extent with CO. Cytochromes cH and L of M. methylotrophus differed from those from Pseudomonas AM1 in having unusually high midpoint redox potentials for non-photosynthetic bacteria and in exhibiting a split alpha-band at low temperatures.
Synthesis of apoprotein B of very low density lipoprotein in embryonic-chick liver. Development of responsiveness of oestrogen
Authors: Nadin-Davis S A , Lazier C B , Capony F , Williams D L
Published: August 2015

The influence of exogenous oestrogen on the rate of synthesis of the apoprotein B of very-low-density lipoprotein by embryonic chick liver has been examined at various stages of development. Significant synthesis of apoprotein B was found in the absence of hormone treatment as early as day 6 of egg incubation. This basal level of apoprotein B synthesis varied from 2.5–6% of total protein synthesis. Embryos at day 10 or earlier treated with oestradiol exhibited no increase in the basal level of apoprotein B synthesis. Embryos at day 11 responded to oestradiol slightly and, from days 12 to 20, oestrogen treatment raised the relative rate of apoprotein B synthesis significantly above basal values: the maximal rate was about 16% of total protein synthesis. The anti-oestrogen tamoxifen citrate abolished the hormone-induced increase in apoprotein B synthesis and was not itself oestrogenic. The basal level of apoprotein B production was not sensitive to tamoxifen citrate, either in relatively early or in later stages of development. The basal level of apoprotein B synthesis, therefore, is oestrogen-independent and under developmental control distinct from the hormone-sensitive synthesis. The ontogeny of oestrogen-responsiveness of apoprotein B production appears to parallel the acquisition of the hepatic oestrogen-receptor system [Lazier (1978) Biochem. J. 174, 143–152].
Studies on the mechanisms of biliary excretion of circulating glycoproteins. The carcinoembryonic antigen
Authors: Thomas P
Published: August 2015

The transport of carcinoembryonic antigen from the circulation of the bile has been studied in the rat. Biliary excretion was directly proportional to the intravenously administered dose. Approximately 1-1.5% of the injected is excreted in the bile. Asialo-(carcinoembryonic antigen), asialo-fetuin and asialo-(alpha 1-acid glycoprotein) behaved similarly. The transit time through the liver for both native- and asialo-(carcinoembryonic antigen) was calculated as 47 min. Sialic acid content was not affected during biliary excretion. Glycoproteins that entered the liver cell lysosomes were not excreted in ther bile. A mechanism by which circulating proteins may by-pass the hepatocytes before being excreted in bile is suggested. Alternative mechanisms of protein entry into bile are discussed.
Inactivation of aspartyl proteinases by butane-2,3-dione. Modification of tryptophan and tyrosine residues and evidence against reaction of arginine residues
Authors: Gripon J C , Hofmann T
Published: August 2015

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.
Human plasma kallikrein. A rapid purification method with high yield
Authors: Nagase H , Barrett A J
Published: August 2015

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).
Poly(adenylic acid) in small amounts, free or covalently linked to substrate, protects RNA from hydrolysis by ribonuclease
Authors: Karpetsky T P , Shriver K K , Levy C C
Published: August 2015

Short lengths (18 residues) of poly(A), covalently linked to the 3′-termini of Escherichia coli 5 S rRNA, induce powerful inhibitions (38-87%) of the activities of RNAases (ribonucleases) from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma. As the polypurine chain length is extended, enzyme activity declines. Furthermore, poly(A) sequences, present only on a small subpopulation of RNA, and accounting for less than 1% of total RNA, serve to protect all RNA, polyadenylated or not, from enzyme-catalysed degradation. The quantity of 3′-terminal adenylic acid residues, relative to the amount of substrate, determines enzyme activity. The exact distribution of a fixed amount of poly(A) residues on the 3′-termini of substrate molecules is unimportant in this respect. Comparison of the efficacies of inhibition of RNAase activity, by using linked poly(A) and similar quantities of free poly(A), revealed that although the free polypurine inhibits RNAase activity, covalent linkage of poly(A) to RNA is more advantageous to the stability of an RNA substrate. However, the ratio of inhibited activities obtained by using linked or free poly(A) may change considerably with alterations in either substrate concentration or polyadenylic acid segment length.
Biophysical characterization of Artemia salina (L.) extracellular haemoglobins
Authors: Wood E J , Barker C , Moens L , Jacob W , Heip J , Kondo M
Published: August 2015

Sedimentation coefficients (s0 20,w) of 11.57 +/- 0.10 S and 11.52 +/- 0.09 S were assigned for Artemia salina (L.) extracellular haemoglobins II and III respectively. These values are not significantly different. The molecular weights, M0w and M0z, of the native haemoglobins as determined by the high-speed sedimentation-equilibrium method were for haemoglobin II 239 400 +/- 7200 and 240 400 +/- 2600 respectively, and for haemoglobin III 216 300 +/- 6500 and 219 300 +/- 4500 respectively. The observed increase of Mapp. with concentration suggested that association was occurring over the concentration range investigated. Exposure of haemoglobin II to either 6 M-guanidinium chloride or to low pH (pH 4) resulted in dissociation to units of approximately half the size of the native protein, with molecular weights approx. 115 000. Electron-microscopic observations indicated a molecular structure composed of two stacked lobed discs. These results strongly support the dimeric model for Artemia haemoglobins proposed by Moens & Kondo [(1978) Eur. J. Biochem. 82, 65-72].
Purification and properties of d-ribulokinase and d-xylulokinase from Klebsiella aerogenes
Authors: Neuberger M S , Hartley B S , Walker J E
Published: August 2015

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.
Immunoelectrophoretic studies on human small-intestinal brush-border proteins
Authors: Skovbjerg H
Published: August 2015

The amounts of lactase (beta-D-galactosidase, EC 3.2.1.23), sucrase (sucrose alpha-D-glucohydrolase, EC 3.2.1.48), maltase (alpha-D-glucosidase, EC 3.2.1.20) microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) in tangentially sectioned biopsies from jejunum were studied by quantitative immunoelectrophoresis and enzymic assays. All enzymes had their maximum activities near the mid-region of the villi and their lowest activities at the bases of the crypts. The ratio between enzyme activity and immunoreactive protein was constant along the villus-crypt axis. This result is consistent with a continuous brush-border-enzyme synthesis as the enterocytes migrate up the villi.
Actin from pig and rat uterus
Authors: Elce J S , Elbrecht A S U , Middlestadt M U , McIntyre E J , Anderson P J
Published: August 2015

Smooth-muscle actin was isolated from pig uterus and from pregnant-rat uterus. Methods involving acetone-dried powders were unsuccessful, and a column-chromatographic procedure was developed, with proteinase inhibitors and avoiding polymerization as a purification step. The yield of pure actin was 0.8–1.5 mg/g wet wt. of uterus, which should be compared with an expected yield of actin from skeletal muscle of 2–4 mg/g wet wt. The actin was pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and exhibited alpha-, beta-, and gamma-forms on isoelectric focusing. It possessed a blocked N-terminal amino acid residue, and its amino acid analysis conformed to those of other actins. The rat uterine actin was available only in small amounts (5–10 mg) and did not polymerize. The pig uterine actin could be obtained in amounts up to 30 mg, polymerized reversibly, and activated a skeletal myosin Mg2+-dependent ATPase.
Tritiation of α-bungarotoxin with N-succinimidyl [2,3-3H]propionate. A useful reagent for labelling proteins
Authors: Dolly J O , Nockles E A V , Lo M M S , Barnard E A
Published: August 2015

Mono[3H]propionyl-alpha-bungarotoxin, prepared with N-succinimidyl [2,3-3H]propionate (sp. radioactivity 50 Ci/mmol) and purified to homogeneity by electrofocusing, retains its biological activity and stability. Rate constants for its binding to acetylcholine receptor were 4.4-fold lower than for unlabelled toxin; no dissociation was detectable. Analysis of enzymic digests of toxin showed 3H is located mainly of entirely in epsilon-propionyl-lysine.