bluuebunny/crossref_metadata_2025 huggingface dataset:
The purification and characterization of bovine C4, the fourth component of complement
Authors: Booth N A , Campbell R D , Fothergill J E
Published: August 2015

The fourth component of complement, C4, was isolated from bovine plasma in high yield, by using simple purification techniques. The protein, like human component C4, is a beta-globulin with a mol.wt. of about 200 000 and consists of three polypeptide chains, alpha, beta and gamma, with apparent mol. wts. of 98 000, 82 000 and 32 000 respectively. The chains of C4 have been separated by methods previously used for human C4. Their amino acid compositions are very similar to those of the human component, but differences in carbohydrate distribution have been observed. The haemolytic activity of bovine C4 is totally destroyed by incubation with bovine C1s, the activated subcomponent of the first component of complement. Component C4, treated in this way, was shown to be cleaved in the alpha chain, which was decreased in mol.wt. by about 9000, corresponding to the removal of subcomponent C4a.
Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy
Authors: Yngner Torsten , Engelbrecht Claes , Lewan Lillemor , Annerfeldt Jan-Erik
Published: August 2015

The balance between anabolism and catabolism of [5-3H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.
Adenosine, thyroid status and regulation of lipolysis
Authors: Ohisalo J J , Stouffer J E
Published: August 2015

Adipocytes from hypothyroid rats do not respond to adrenaline with increased glycerol release. Adenosine deaminase largely restores lipolytic sensitivity. This effect is reversed by 2-deoxycoformycin, an inhibitor of the enzyme, and by N6-(phenylisopropyl)adenosine, which is not deaminated. Lipolytic response of normal cells to adrenaline is only 50% inhibited by phenylisopropyladenosine, whereas in cells from hypothyroid rats blockage is total. Inhibition of 50% was seen at 100 and 1 nM concentrations respectively. Insensitivity to adrenaline of hypothyroid-rat adipocytes can, at least partly, be explained by increased sensitivity to adenosine.
Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform
Authors: Tyler D. D. , Webb Pauline R.
Published: August 2015

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60–70μmol/min per mg of protein by (NH4)2SO4 fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their Km values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1–2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533–537] is similar to other preparations described previously and that this method is superior in simplicity and speed.
Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo
Authors: Majamaa Kari , Kuutti-Savolainen Eeva-Riitta , Tuderman Leena , Kivirikko Kari I.
Published: August 2015

The turnover of prolyl hydroxylase and an immunoreactive protein that corresponds in size to the smaller subunit of the enzyme was studied in vivo after injection of [3H]leucine into 11-day chick embryos. The specific radioactivity and total radioactivity of the monomer-size protein were much higher than those of the enzyme tetramers in the cartilaginous bone at 3h and 12h after the radioisotope injection, indicating that the monomer-size protein represents precursors rather than degradation products of the enzyme tetramers. Between 24 and 144h after the injection the specific radioactivity and total radioactivity of the two forms of the enzyme protein showed essentially identical decay rates, the observed specific radioactivity of the monomer-size protein being about 120–130% and total radioactivity about 80% of that of the enzyme tetramers. The true half-life, when corrected for dilution caused by tissue growth and re-utilization of the [3H]leucine, was 37.9h for the monomer-size protein and 39.0h for the tetramers. The results obtained in the lung were less reliable owing to high blank radioactivity values in the immunoprecipitation, but even so some definite differences were found between this tissue and the cartilaginous bone. The specific radioactivity of both forms of the enzyme protein at 24h was only about 20–25% of that in the cartilaginous bone. The total radioactivity of the monomer-size protein in the lung remained about 5 times that of the enzyme tetramers, whereas it was only about 0.8 times that of the tetramers in the cartilaginous bone. As in the cartilaginous bone, the decay rates of both forms of the enzyme protein were essentially identical in the lung, with a true half-life of about 46h. The results suggest that the rate of prolyl hydroxylase synthesis is slower in the lung than in the cartilaginous bone, whereas the degradation rates are fairly similar in these two tissues. The data further suggest that, in the lung at least, a large part of the monomer-size protein became degraded without being converted into enzyme tetramers.
Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate
Authors: Taylor Wayne M. , Halperin Mitchell L.
Published: August 2015

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-14C]glucose into 14CO2. Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into 14CO2. Apparent rates of glucose transport were significantly increased by insulin, H2O2, adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H2O2, adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H2O2, adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.
Polyamine replacement by magnesium ions in BHK-21/C13 cells
Authors: Melvin Maureen A. L. , Keir Hamish M.
Published: August 2015

Cultures of BHK-21/C13 cells, whose growth was inhibited by deprivation of serum, were stimulated to grow by addition of serum to the culture medium. Addition of MgCl2 to the medium, to increase the concentration of Mg2+ ions by 15mm, 30min before addition of serum, had no effect on the stimulation of cell growth, but inhibited the accumulation of cellular spermidine, so that the spermidine/spermine molar ratio was lower in these cultures than in cultures that had received no additional cations. The increase in the activity of ornithine decarboxylase that occurs 4–5h after serum ‘step-up’ was substantially diminished by increasing the concentration of Mg2+ ions, but not of Na+ or K+ ions, in the medium by 30mm, 30min before addition of serum, and this inhibition was maintained for at least 24h. Methylglyoxal bis(guanylhydrazone), added to serum-deprived cultures to a concentration of 20μm, 30min before addition of serum, severely inhibited the increase in cell growth. The inhibitory effects of the drug were prevented by simultaneous addition of spermidine to the medium (to 100μm), and were partly prevented by the simultaneous addition of Mg2+ ions (to 30mm). Mg2+ ions were particularly effective in overcoming the inhibitory effect of methylglyoxal bis(guanylhydrazone) on the synthesis of DNA. Thus although a certain lack of specificity for cations exists in BHK-21/C13 cells, in that Mg2+ ions can be substituted for polyamines, particularly spermidine, to some extent, there are cellular processes for which the requirement for polyamines as cations is specific.
Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium
Authors: Ibbotson G E , Williams K E
Published: August 2015

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.
Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins
Authors: DeFilippi Louis J. , Toler Linda S. , Hultquist Donald E.
Published: August 2015

The reactivities of alkaline NH2OH and neutral NaHSO3 with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2–3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH2OH and neutral NaHSO3. The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH2OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH2OH, but not with neutral NaHSO3. These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH2OH or NaHSO3, but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927–3932] may have been premature. These studies also provide evidence that the α-hydroxyfarnesylethyl side chain of haem a affects the α-band maximum, but not the β- or Soret bands of the reduced pyridine haemochrome spectrum of haem a.
The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c
Authors: Wallace C J , Offord R E
Published: August 2015

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.
Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy
Authors: Maroun L E
Published: August 2015

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.
Studies on horseradish peroxidase in dimethyl sulphoxide/water mixtures. The activation of hydrogen peroxide and the binding of fluoride
Authors: Adams P A , Baldwin D A , Collier G S , Pratt J M
Published: August 2015

We studied the variation in spectra and in reactivity towards H2O2 of solutions of horseradish peroxidase in dimethyl sulphoxide/water mixtures, obtained by diluting stock solutions of the enzyme in either water or dimethyl sulphoxide, and assayed the enzyme activity and studied the binding of F- by the peroxidase in 65% (v/v) dimethyl sulphoxide. A broadly similar pattern of changes is observed whether one starts from water or from dimethyl sulphoxide; the changes are essentially reversible, though hysteresis is observed. When the dimethyl sulphoxide content of the solvent mixture is increased, the peroxidase retains its ability to activate H2O2 up to 74% (v/v) dimethyl sulphoxide. The peroxidase in 65% (v/v) dimethyl sulphoxide binds F- together with a proton (or the equivalent loss of HO-), as already established for aqueous solutions. We point out that the occurrence in such solutions of both the ability to activate H2O2 and the inability to bind F- without taking up H+ or losing HO- supports the proposed mechanism for activating H202, whereby the protein binds the substrate in the form of the much more reactive HO2-.
The amino acid sequence of ferredoxin from Triticum aestivum (wheat)
Authors: Takruri I , Boulter D
Published: August 2015

The amino acid sequence of the ferredoxin of Triticum aestivum (wheat) was determined by using a Beckman 890C sequencer in combination with the dansyl–phenylisothiocyanate method to characterize peptides obtained by tryptic, chymotryptic and thermolytic digestion of CNBr-cleavage fragments. The molecule consists of a single polypeptide chain of 97 residues and has an unblocked N-terminus. It shows considerable similarity to other plant-type ferredoxins.
Are polyphosphoinositides associated with glycophorin in human erythrocyte membranes?
Authors: Shukla S D , Coleman R , Finean J B , Michell R H
Published: August 2015

Glycophorin prepared by a lithium di-iodosalicylate-extraction/phenol-partition method was rich in polyphosphoinositides (phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate), but glycophorin extracted by Triton X-100 showed no such enrichment. The enrichment observed in the former preparations appeared not to be caused by pre-existing association between glycophorin and polyphosphoinositides in the human erythrocyte membrane, but to be largely a consequence of the preparative procedures.
Kinetics of enzymic reductive deiodination of iodothyronines. Effect of pH
Authors: Visser T J , Fekkes D , Docter R , Hennemann G
Published: August 2015

5′-Deiodination of thyroxine (yielding 3,3′,5-tri-iodothyronine; reaction I) and of 3,3′,5′-tri-iodothyronine (yielding 3,3′-di-iodothyronine; reaction II) and 5-deiodination of thyroxine (yielding 3,3′,5′-tri-iodothyronine; reaction III) and of 3,3′,5-tri-iodothyronine (yielding 3,3′-di-iodothyronine; reaction IV) as catalysed by rat liver microsomal fraction were studied at pH 6.5, 7.2 and 8.0 It was found that: (1) the Km of reaction I was relatively independent of pH (approx. 3 microM), whereas V was highest at pH 6.5 (63 pmol of 3,3′,5-tri-iodothyronine/min per mg of protein); (2) the Km of reaction II was lowest at pH 6.5 (0.035 microM), but V was highest at pH 8.0 (829 pmol of 3,3′-di-iodothyronine/min per mg of protein); (3) thyroxine inhibited reaction II competitively; Ki values were identical at pH 6.5 and 8.0 (1 microM); (4) for both reactions III and IV Km was lowest and V was highest at pH 8.0. The results are compatible with the view that reactions I and II are mediated by a single enzyme (iodothyronine 5′-deiodinase) and that reactions III and IV are catalysed by a second enzyme (iodothyronine 5-deiodinase).
Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain
Authors: Knauer Thomas E.
Published: August 2015

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10μm-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl2, and 5,5′-dithiobis-(2-nitrobenzoic acid), and exhibit the same Km (1.8μm) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0°C), but there is no change in the Km with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10μm-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.
Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I
Authors: Rohde H , Wachter E , Richter W J , Bruckner P , Helles O , Timpl R
Published: August 2015

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7–8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.
Structure of antigenic determinants in the N-terminal region of dermatosparactic sheep procollagen type I
Authors: Rohde H , Timpl R
Published: August 2015

About half of the rabbit antisera raised against type-I procollagen, p alpha 1(I) chain or nonreduced procollagen peptides reacted in a radioimmunoassay with the reduced form of peptide Col 1, which comprises the whole non-collagenous region at the N-terminus of procollagen. Proteolytic fragments prepared from reduced peptide Col 1 were still effective inhibitors of the antibodies and allowed the localization of two antigenic determinants. The antigenically active regions have the sequences less than Glu-Glu-Glu-Gly-Gln-Gln-Glu and Gly-Asp-Thr-Gly-Pro-Arg, and are located at the N- and C-termini of the peptide respectively. Antibodies raised against reduced peptide Col 1 bind to a determinant localized in a different region of the peptide.
The reaction of menadione with haemoglobin. Mechanism and effect of superoxide dismutase
Authors: Winterbourn C C , French J K , Claridge R F
Published: August 2015

1. Menadione was found to react with both the haem groups and the beta-93 thiol groups of haemoglobin. 2. It oxidized the haem groups of oxyhaemoglobin, giving mainly methaemoglobin and a smaller amount of haemichrome. The reaction rate was decrease in the presence of catalase and markedly accelerated in the presence of superoxide dismutase. It is proposed that the overall reaction involves the initial reversible formation of methaemoglobin and the semiquinone, and that the effect of superoxide dismutase is to prevent the reverse reaction, by removing superoxide and hene O2-. E.s.r. evidence for the information of the semi-quinone and its reactions is presented. 3. The reaction of menadione with the beta-93 thiol groups of haemoglobin appeared to be similar to that with other thiols, forming the 3-thioether derivative of menadione, but it was also accompanied by reduction of methaemoglobin. This reduction was prevented by superoxide dismutase, but appeared to be caused by the semiquinone radical, which was produced as an intermediate. 4. Reduced glutathione functioned only to a limited extent as a scavenger of the menadione semiquinone. Its main reaction was directly with menadione to form the thioether. Ascorbate was a more efficient scavenger, and accelerated the oxidation of oxyhaemoglobin by menadione. 5. The significance of these findings in relation to menadione-induced erythrocyte haemolysis is discussed.
Regulation of ketogenesis during the suckling–weanling transition in the rat. Studies with isolated hepatocytes
Authors: Benito M , Whitelaw E , Williamson D H
Published: August 2015

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175–E180].
Taurine transport in renal brush-border-membrane vesicles
Authors: Rozen R , Tenenhouse H S , Scriver C R
Published: August 2015

Taurine transport in isolated brush-border-membrane vesicles from rat kidney is concentrative and it is driven by the Na+ gradient and transmembrane potential difference; binding is not a significant component of net uptake. The Na+-dependent component of net uptake is saturable with an apparent Km of 17 microM. The taurine-transport mechanism is selective for beta-amino compounds.
Induction of liver cell haem oxygenase in iron-overloaded rats
Authors: Ibrahim N G , Hoffstein S T , Freedman M L
Published: August 2015

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.
Adrenaline responsiveness of glucose metabolism in insulin-resistant adipose tissue of rats fed a high-fat diet
Authors: Susini C , Lavau M , Herzog J
Published: August 2015

The effects of adrenaline (0.5 microM) and the combination of adrenaline and insulin (1.7nM) on [6-14C]glucose metabolism were assessed in epididymal fat-pads from rats fed either a low- or high-fat diet. The response of lipolysis to adrenaline was clearly diminished in fat-fed rats. Insulin added to adrenaline inhibited the lipolysis by 50% regardless of the diet. Glucose utilization in adipose tissue of fat-fed rats was markedly stimulated by adrenaline (glucose uptake was increased 3-fold and the production of CO2 and the glycerol moiety of acylglycerol was increased 4-fold). However, adipose tissue from fat-fed rats was resistant to the effect of insulin to produce a further increase in adrenaline-stimulated glucose uptake. The intracellular capacity of lipogenesis on the one hand, and the production of CO2 and the glycerol moiety of acylglycerol on the other, are of prime importance in the action of insulin and adrenaline on glucose utilization in this model.
Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles
Authors: Stoll Reinhard , Kinne Rolf , Murer Heini
Published: August 2015

Renal brush-border membrane vesicles isolated from rats kept for 6–8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na+-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with Pi (5ml of 20mm-NaH2PO4) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na+/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na+/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.
Hormonal control of uteroglobin secretion in rabbit uterus. Inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration
Authors: Kopu Helena T. , Hemminki Seija M. , Torkkeli Tuula K. , Jänne Olli A.
Published: August 2015

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5α-dihydrotestosterone, either alone or concomitantly with oestradiol-17β and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)–cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5α-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17β (50μg/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5α-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17β or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17β (50μg/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.
Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin
Authors: Denton R M , Edgell N J , Bridges B J , Poole G P
Published: August 2015

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.
Relationship between phosphatidylinositol synthesis and recovery of 5-hydroxytryptamine-responsive Ca2+ flux in blowfly salivary glands
Authors: Fain J N , Berridge M J
Published: August 2015

Each salivary gland contains about 135 pmol of phosphatidylinositol. In glands prelabelled by incubation for 1 h with [32P]Pi or [3H]inositol there was a subsequent breakdown of 80% of the labelled phosphatidylinositol over a 2 h incubation period with 10 micrometer-5-hydroxytryptamine. However, there was no detectable decrease either in total phosphatidylinositol based on phosphorus analysis by chemical estimation or in the radioactivity of [32P]phosphatidylinositol in salivary glands of flies raised from the larval stage on diets containing[32P]Pi and whose phospholipids were uniformly labelled. These results suggest that the pool of phosphatidylinositol involved with Ca2+ gating is a small fraction of the total phosphatidylinositol content. Furthermore it is this small compartment that is preferentially radioactively labelled during short-term incubations with radioactively labelled precursors. In salivary glands incubated for 2 h with 10 micrometer-5-hydroxytryptamine there was a marked decrease in the flux of 45Ca2+ across the gland. After removal of the hormone, incubation of salivary glands for 1 h in the presence of 2mM-inositol, but not choline or ethanolamine, resulted in a recovery of hormone-responsive 45Ca2+ flux. Quantitative studies revealed that less than 9 pmol of phosphatidylinositol must be formed to fully restoret he 5-hydroxytryptamine-responsive 45Ca2+ flux.
Lipoprotein lipase activity of rat cardiac muscle. The intracellular distribution of the enzyme between fractions prepared from cardiac muscle and cells isolated from the hearts of fed and starved animals
Authors: Chohan P , Cryer A
Published: August 2015

1. Subcellular fractions, characterized by using morphological, compositional and enzymic markers, were prepared from rat heart tissue and cells isolated from the hearts of fed and 24 h-starved rats. 2. The lipoprotein lipase activity of fractions from whole tissue and isolated cells was determined in either fresh fractions or in acetone/diethyl ether powders of the fractions. 3. Lipoprotein lipase activity was present in all the fractions from tissue and cells, but was found to be of highest relative specific activity in the microsomal () fractions. 4. In fractions prepared from the isolated cells of hearts from starved rats the proportion of the total lipoprotein lipase present and its relative specific activity in the microsomal fraction were greater than in the equivalent fractions from fed animals. 5. The enhancement of lipoprotein lipase activity as a result of the acetone/diethyl ether powder preparation of fractions was most extensive in the microsomal fractions. 6. Investigation of the microsomal fraction showed that the lipoprotein lipase activity present was in two pools, one of which was within endoplasmic-reticulum vesicles. 7. The observations were consistent with the possibility that the cardiac-muscle cell could be the origin of the lipoprotein lipase activity functional in triacylglycerol uptake by the heart.
Some molecular properties of NAD(P)H dehydrogenase from rat liver
Authors: Wallin Reidar
Published: August 2015

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same Km value for NADH but different values for Vmax. were obtained for the enzyme purified from Sprague–Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.
Steady-state investigations of the mechanism of histidinol dehydrogenase
Authors: Görisch H
Published: August 2015

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.
Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria
Authors: Castroviejo M , Tharaud D , Tarrago-Litvak L , Litvak S
Published: August 2015

Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3′- or 5′-labelled polydeoxyribonucleotides, as well as a ‘proof-reading’ capacity.
Biosynthesis of proline in Pseudomonas aeruginosa. Properties of ;-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase
Authors: Krishna Rangachar V. , Beilstein Paul , Leisinger Thomas
Published: August 2015

γ-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from γ-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)+. The apparent Km values were: NAD+, 0.36mm; NADP+, 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP+ and 8mm with NAD+; Pi, 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of γ-glutamyl phosphate, inhibited this enzyme competitively (Ki=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH4)2SO4 fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (Km=0.05mm) was a better substrate than NADPH (Km=0.02mm). The apparent Km values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD+ at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)+, heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.
Dansylation of human serum albumin in the study of the primary binding sites of bilirubin and l-tryptophan
Authors: Jacobsen C , Jacobsen J
Published: August 2015

Binding of bilirubin and of L-tryptophan to dansylated albumins was investigated. Dansylation of less than one lysine residue per molecule of albumin did not affect the bilirubin binding, but decreased the L-tryptophan binding, indicating that dansylation had taken place in or near the l-tryptophan-binding site. Native albumin and albumin-bilirubin 1:1 complex showed the same affinity for L-tryptophan. The results indicate that, although L-tryptophan and bilirubin are bound in the same region, perhaps in a common cavity of the albumin molecule, such a cavity is sufficiently large to contain both ligands.
Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria
Authors: Choo K H , Cotton R G H , Danks D M , Jennings I G
Published: August 2015

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.