bluuebunny/crossref_metadata_2025 huggingface dataset:
The enzymic degradation of l-serine O-sulphate by a specific system from pig liver. Studies on the mechanism of the reaction
Authors: Tudball N. , Thomas P.
Published: August 2015

1. By using [18O]water it was demonstrated that l-serine O-sulphate undergoes C–O cleavage during enzyme-catalysed degradations. 2. C–O cleavage of substrate also occurs under the agency of alkali and pyridoxal phosphate, but, as expected, acid-catalysed degradation involves S–O scission. 3. Aminoacrylate was identified as an intermediate in the enzyme-catalysed degradation of l-serine O-sulphate.
Recovery of steroidal glucosiduronic acids from organic solvents containing anionic liquid ion-exchangers
Authors: Mattox Vernon R. , Litwiller Robert D. , Goodrich June E.
Published: August 2015

Solutions of anionic liquid ion-exchangers in organic solvents are potentially useful for extracting steroidal glucosiduronic acids from biological fluids and for purifying mixtures of these acids by chromatography. If a glucosiduronic acid is to be isolated in pure form after either of these procedures, it is necessary to separate it from the ion-exchanger. Separation from an organic solution of tetraheptylammonium chloride may be accomplished by extraction with water under the following conditions, which promote transfer of a glucosiduronate to the aqueous phase: (1) an appropriate solvent (diluent) as the organic phase, (2) the presence in the two-phase mixture of an anion such as myristate or dodecyl sulphate to combine with the tetraheptylammonium ion, and (3) an increase of the pH of the aqueous phase in association with the presence of myristate or dodecyl sulphate. The foregoing factors apply also to removal of glucosiduronates from organic solutions of ion exchangers that are hydrochlorides of tertiary, secondary, or primary amines. Since these amines exert progressively less solubilizing effect for glucosiduronates as the pH of the aqueous phase is increased, the conjugates can be released from the organic phase by adjusting the pH to 10 and omitting the myristate or dodecyl sulphate.
Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation
Authors: Hardingham T. E. , Muir Helen
Published: August 2015

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.
Soluble adenosine diphosphate glucose–α-1,4-glucan α-4-glucosyltransferases from spinach leaves
Authors: Ozbun J. L. , Hawker J. S. , Preiss Jack
Published: August 2015

Multiple forms of ADP-glucose–α-1,4-glucan α-4-glucosyltransferase were obtained from spinach leaves by gradient elution from a DEAE-cellulose column. In the presence of high concentrations of some salts and bovine serum albumin, unprimed activity was found in one (transglucosylase III) of the four fractions eluted from the column. In addition to having unprimed activity, transglucosylase III had a lower Km for ADP-glucose, a much higher Km for oyster glycogen, greater heat sensitivity and lower affinity for maltose, maltotriose and amylopectin β-limit dextrin than fractions I, II and IV. In addition, the kinetics at low concentrations of amylose, amylopectin and rabbit liver glycogen were non-linear for transglucosylase III. The properties of transglucosylases I, II and IV were generally similar to each other. Rates of the unprimed reaction at physiological concentrations of ADP-glucose were greater than those found for the primed reaction of fraction III. The product formed by the unprimed reaction was a glucan containing principally α-1,4 linkages with some α-1,6 linkages. The primer, maltose, at a concentration of 0.5m inhibited the synthesis of the unprimed product.
Hysteresis and conformational changes in ribosomal ribonucleic acid
Authors: Cox R. A. , Katchalsky A.
Published: August 2015

Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3–7 at approx. 0°C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ‘scanned’, e.g. by titration from pH7 to a point in the range pH3–4 followed by titration with alkali to pH7. It is inferred that the ‘lag’ in the release of certain bound protons is at least 1 pH unit, that at least about 9–15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ‘scanning’ curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A)·poly(AαH+)-like regions in acidic solutions. It is also inferred that the extent of ‘hysteresis’ might depend on the primary nucleotide sequence of rRNA as well as on secondary structure.
The initial synthesis of proteins during development. Phosphoenolpyruvate carboxylase in rat liver at birth
Authors: Philippidis Helen , Hanson R. W. , Reshef Lea , Hopgood M. F. , Ballard F. J.
Published: August 2015

1. A specific antibody, prepared by immunizing rabbits with phosphoenolpyruvate carboxylase (EC 4.1.1.32) purified from adult rat liver, was used to study the appearance of this enzyme in livers from developing rats. 2. Although some inactive precursor of the enzyme may be present in foetal liver, the amount is not sufficient to account for the enzyme appearance at birth. 3. The rate of phosphoenolpyruvate carboxylase synthesis relative to other cytosol proteins increases 20-fold from the foetus to the 1-day-old rat. The high rate of synthesis was maintained at least until 3 days after birth. 4. There was no measurable degradation of phosphoenolpyruvate carboxylase during the first day after birth. During this period the hepatic enzyme content increased 12-fold. 5. When phosphoenolpyruvate carboxylase attained a constant activity in the liver of rats 2 days after birth the half-time of degradation was approx. 13h. 6. We suggest that the pattern of changes occurring during appearance of phosphoenolpyruvate carboxylase is similar to substrate-induced enzyme induction in bacteria.
Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices
Authors: Lapetina Eduardo G. , Michell Robert H.
Published: August 2015

1. Rat cerebral-cortex slices were incubated with 32Pi, acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.
Polyprenol phosphate as an acceptor of mannose from guanosine diphosphate mannose in Aspergillus niger
Authors: Barr R. M. , Hemming F. W.
Published: August 2015

Growth of Aspergillus niger in the presence of [2-14C]mevalonate and 32Pi led to the formation of a lipid, containing 14C (0.14% of dose) and 32P (0.009% of dose), that had chromatographic properties identical with those of exo-methylene-hexahydropolyprenol phosphate. When a particulate enzyme preparation from the thallus of A. niger was incubated with GDP-[14C]mannose, the main radioactive products were mannose 1-phosphate (57% of products) and mannose (18%). In addition radioactive mannan (8%) and a mannolipid (2%) were formed. The latter was identified as exo-methylene-hexahydropolyprenol phosphate mannose on the basis of its chromatographic properties, acid lability and on the increase in formation of the mannolipid when the phosphate of exo-methylene-hexahydropolyprenols was added to the incubation mixture. The phosphates of ficaprenols and cetyl alcohol caused no corresponding increase. These observations are interpreted as evidence that the thallus of A. niger contains a mannose transferase that uses the phosphate of exo-methylene-hexahydropolyprenols as an acceptor. This situation is discussed in the light of the analogous involvement of a prenol phosphate mannose as an intermediate in the biosynthesis of bacterial wall mannan.
Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The interaction of cholesteryl 14-methylhexadecanoate
Authors: Hradec J.
Published: August 2015

1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11–26% and the simultaneous binding of GTP to 32–43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA–GTP–transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.
Cholesterol 7α-hydroxylase in rat liver microsomal preparations
Authors: Mitropoulos K. A. , Balasubramaniam S.
Published: August 2015

Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7α-hydroxycholesterol, 7-oxocholesterol, 7β-hydroxycholesterol and 5α-cholestane-3β,5,6β-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-14C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7α-hydroxy[14C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7α-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7α-hydroxylase and to measure low concentrations of endogenous 7α-hydroxycholesterol in liver.
Purification and characterization of lysosomal β-glucuronidase from human placenta
Authors: Contractor S. F. , Shane B.
Published: August 2015

1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. β-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl β-d-glucuronide and phenolphthalein β-d-glucuronide as substrates. 4. Only one isoenzyme of β-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated β-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental β-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma β-glucuronidase was also present in plasma.
Biomethylation of deoxyribonucleic acid in cultured human tumour cells (HeLa). Methylated bases other than 5-methylcytosine not detected
Authors: Lawley P. D. , Crathorn A. R. , Shah S. A. , Smith B. A.
Published: August 2015

1. Incorporation of methyl groups from [methyl-14C]methionine into DNA of dividing HeLa cells was investigated, essentially by the procedures of Culp, et al. (1970). 2. Contrary to the report of the latter, but in agreement with other work on biomethylation of mammalian DNA, 5-methylcytosine was the sole methylated base detected. 3. Chromatographic separations of 3-methylcytosine from 5-methylcytosine and purines are discussed.
The specific assay of arylsulphatase C, a rat liver microsomal marker enzyme
Authors: Milsom D. W. , Rose F. A. , Dodgson K. S.
Published: August 2015

Conditions based on previous assays with potassium p-acetylphenyl sulphate have been established for the specific assay of arylsulphatase C in rat tissues. The enzyme has optimum activity with 40mm substrate at pH8.0 in the presence of 0.1m-phosphate buffer. Under these conditions arylsulphatase C can be assayed without interference from the other arylsulphatase enzymes present and is useful as a marker for the endoplasmic reticulum in cell-fractionation studies.
Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts of Euglena gracilis
Authors: Avadhani N. G. , Buetow D. E.
Published: August 2015

1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.
The transformation of the cytoplasmic oestradiol–receptor complex into the nuclear complex in a uterine cell-free system
Authors: Gschwendt Michael , Hamilton Terrell H.
Published: August 2015

Experiments performed with a cell-free system in tris–EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol–receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2°C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37°C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37°C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.
Adenosine 5′-pyrophosphate sulphurylase in baker's yeast
Authors: Adams C. A. , Nicholas D. J. D.
Published: August 2015

ADP sulphurylase from baker's yeast was purified and its properties were studied. The enzyme is very heat-labile and its activity shows linear kinetics over narrow ranges of time and protein concentration. It is not activated by metals and is inhibited by thiol-reactive compounds. The enzyme, which replaces inorganic sulphate in adenosine 5′-sulphatophosphate with Pi to yield ADP, also catalyses an exchange of Pi into ADP. Kinetic studies show that the enzyme has a high affinity for adenosine 5′-sulphatophosphate, although concentrations in excess of 1.0mm are inhibitory. However, the kinetics for Pi are more complex and the enzyme is not inhibited by Pi up to 20.0mm.
The osmotic properties of sulphoethyl-Sephadex. A model for cartilage
Authors: Ogston A. G. , Wells J. D.
Published: August 2015

1. Observations are reported on the variation of the swelling of sulphoethyl-Sephadex C-50 and C-25, and of the partition of NaCl between solution and gel, with the concentration of NaCl. 2. The results were interpreted in terms of Manning's (1969) treatment of the interactions between polyionic polymers and simple electrolytes and of Flory's (1953) treatment of the swelling of gels. 3. The results indicate net inner osmotic pressures as high as 3.6×105Pa (140 mosmolar=3.6×105N/m2) in 0.15m-NaCl. 4. It is suggested that cartilage may have net inner osmotic pressure of the order of 105Pa.
The reaction of papain with Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoate) dianion]
Authors: Brocklehurst K. , Kierstan M. , Little G.
Published: August 2015

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs2−) produced by reaction of papain with the 5,5′-dithiobis-(2-nitrobenzoate) dianion (Nbs22−; Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs22− at pH8, Nbs22− reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs2− in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain–Nbs−. 3. Papain catalyses the hydrolysis of Nbs22− at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.
Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue
Authors: Kanoh H. , Lindsay D. B.
Published: August 2015

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-14C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C18 and C20 fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Δ11:12 isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Δ11:12 isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C16 and C18 monoenoic acids; synthesis of C20 acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.
Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases
Authors: Holbrook J. J. , Yates D. W. , Reynolds S. J. , Evans R. W. , Greenwood C. , Gore M. G.
Published: August 2015

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (α) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to α for NADH binding to give the alcohol dehydrogenase–NADH–isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase–NADH complex and the bovine glutamate dehydrogenase–NADH–glutamate complex. The non-linearity is accurately represented by F=[1−α(1−x)]n where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and α. 4. The prediction from the equation F=[1−α(1−x)]n that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when α=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.
The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli
Authors: Gray W. J. H. , Midgley J. E. M.
Published: August 2015

The biosynthesis and stability of various RNA fractions was studied in RCstr and RCrel multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RCstr mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA–RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60–70% of the labelled RNA formed in RCstr mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RCrel strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RCrel strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RCstr or RCrel strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RCstr auxotrophs was unchanged from that found in normally growing cells. In contrast, RCrel strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.
Guanine deaminase in rat liver and mouse liver and brain
Authors: Kumar K. S , Sitaramayya A. , Krishnan P. S.
Published: August 2015

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis–Menten kinetics. The Km value of enzyme A for guanine was 5.3μm and that of enzyme B 20μm. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the Km values for the reaction with guanine were respectively 5 and 66μm. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.
Stereochemical aspects of the formation of double bonds in abscisic acid
Authors: Milborrow B. V.
Published: August 2015

The stereochemistry of the hydrogen elimination that occurs during the formation of the Δ4- and Δ2′-double bonds of abscisic acid has been determined from the 14C/3H ratios in abscisic acid biosynthesized by avocado fruit from [2-14C,(2R)-2-3H1]-, [2-14C,(2S)-2-3H1]- and [2-14C,(5S)-5-3H1]-mevalonate. Setting the 14C/3H ratio at 3:3 for [2-14C,(2R)-2-3H1]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-14C,(2S)-2-3H1]mevalonate was 3:1.63. Removal of the 3′-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3′-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-14C,(2R)-2-3H1]-mevalonate and to 3:1.05 for [2-14C,(2S)-2-3H1]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one 3H atom at the 6′-methyl position of abscisic acid from the [(2R)-2-3H1]- and [(2S)-2-3H1]-mevalonate. The presence of some label at positions 3′ and 4 when [(2S)-2-3H1]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.
The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin
Authors: Elleman T. C.
Published: August 2015

1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models.
A study of the cell surface of tumour, foetal and lymph-node cells by cell electrophoresis after antibody and enzymic treatment
Authors: Woo Jean , Cater Donald B.
Published: August 2015

1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH–electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.
Norephedrines as metabolites of [14C]amphetamine in urine in man
Authors: Caldwell J. , Dring L. G. , Williams R. T.
Published: August 2015

(±)-[14C]Amphetamine sulphate (20mg) was administered orally to each of two human male subjects, and 80–85% of the14C was excreted in the urine in 2 days. The metabolites in the first day's urine were examined quantitatively. Apart from the metabolites previously described (Dring et al., 1970), norephedrine (2.2 and 2.6% of dose in the two subjects respectively) and 4-hydroxynorephedrine (0.3 and 0.4% of dose in the two subjects respectively) were also found.
Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase
Authors: Hardingham T. E. , Fitton-Jackson Sylvia , Muir Helen
Published: August 2015

Explants of cartilage from tibiae of 11–12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.
A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein
Authors: Mather I. H. , Knight M.
Published: August 2015

A thermostable NAD(P)+glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.
Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth
Authors: Abraham K. A. , Andersen K. J. , Rognes A.
Published: August 2015

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T4 DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T4 DNA.
Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria
Authors: Spencer T. , Bygrave F. L.
Published: August 2015

1. Added Ca2+stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent Km for added Ca2+in stimulating the translocation of 200μm-ATP is approx. 160μm (75μm ‘free’ Ca2+). 3. The greatest stimulation of ATP translocation by Ca2+occurs at the lower concentrations of ATP. 4. Sr2+(and to a lesser extent Ba2+) can replace Ca2+whereas Mg2+and Mn2+have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca2+whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca2+and by uncouplers is additive and that the mechanism of Ca2+stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca2+, ATP is able to effectively compete with ADP for translocation. 8. Added K+further enhances the ability of Ca2+to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca2+in modifying enzymic reactions involved in the regulation of cell metabolism.
Microbial metabolism of alkylbenzene sulphonates. Bacterial metabolism of undecylbenzene-p-sulphonate and dodecylbenzene-p-sulphonate
Authors: Willetts A. J. , Cain R. B.
Published: August 2015

1. A study was made of the biodegradation of alkylbenzene sulphonate homologues, one of the major components of commercially marketed detergents. A Bacillus species was elected for growth on alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. 2. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of alkylbenzene sulphonate homologues were all further metabolized by the Bacillus species. 3. The alkyl side chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidized by a β-oxidation pathway. Three enzymes of the β-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and β-hydroxyacyl-CoA dehydrogenase, were identified in cell-free extracts of the detergent-grown Bacillus species. The substrate specificity of acyl-CoA synthetase indicated activity towards several alkylbenzene sulphonate homologues. 4. The sulphonate moiety was released as sulphite by a desulphonating enzyme. Some kinetic properties of this enzyme were determined. The sulphite was subsequently metabolized to either sulphate or adenosine 5′-sulphatophosphate. Two enzymes involved in sulphite metabolism, i.e. sulphite–cytochrome c reductase and adenosine 5′-sulphatophosphate–cytochrome c reductase were detected in cell-free extracts of undecylbenzene-p-sulphonate-grown Bacillus species. 5. The combined results of continuous sampling programmes monitored by both t.l.c. and sulphite appearance in the growth medium indicated that desulphonation of the aromatic moiety was the likely first step in the overall biodegradation of several alkylbenzene sulphonate homologues. 6. The presence of p-hydroxyphenylpropionate, p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on several alkylbenzene sulphonate homologues containing an odd number of carbon atoms in the side chain was confirmed by g.l.c. and t.l.c. analysis. Cells grown on several homologues containing an even number of carbon atoms in the side chain were shown to contain p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. 7. The aromatic nucleus obtained from undecylbenzene-p-sulphonate was further metabolized by an oxidation sequence involving an ‘ortho-cleavage’ route. 8. An overall metabolic pathway for the biodegradation of various alkylbenzene sulphonate homologues by this Bacillus species is proposed.
Prenatal and postnatal changes in the content and species of ferritin in rat liver
Authors: Linder Maria C. , Moor Joan R. , Scott Laura E. , Munro Hamish N.
Published: August 2015

The iron and ferritin content of rat liver and the species of ferritin present were examined from 4 days before to 3 weeks after birth. 1. Total iron and ferritin iron accumulated rapidly during the last days of gestation and from the second postnatal day underwent a steady depletion. 2. The amount of iron deposited before birth in the liver of each pup varied inversely with litter size and could be increased moderately by injection of iron into the mother before mating. 3. Intraperitoneal injection of iron 1 day after birth doubled the concentration of total iron, ferritin iron and ferritin protein in the liver over the next 24h, but at 3 weeks after birth it raised the very low concentrations of iron and ferritin severalfold. 4. As shown by electrophoretic migration, ferritin and dissociated ferritin subunits prepared from the livers of rats from 4 days before to 3 weeks after birth differed from those of adult liver ferritin and were indistinguishable from those of adult kidney and spleen ferritin. Treatment with iron at 3 weeks of age induced formation of a ferritin with electrophoretic properties resembling those of adult liver. It is concluded that iron given at this stage of development may activate the genetic cistron for adult liver ferritin.
The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena
Authors: Bitar K. G. , Vinogradov S. N. , Nolan C. , Weiss L. J. , Margoliash E.
Published: August 2015

1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70–80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.
Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver
Authors: Heirwegh K. P. M. , Van De Vijver M. , Fevery J.
Published: August 2015

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0°C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of Pi) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis–Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis–Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn2+ was slightly more, and Ca2+ somewhat less, stimulatory than Mg2+. The Mg2+-dependent fraction showed Michaelis–Menten kinetics with respect to the added Mg2+. 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.
Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver
Authors: Fevery J. , Leroy P. , Heirwegh K. P. M.
Published: August 2015

1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6–7.2. Michaelis–Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg2+; Ca2+ was slightly less, and Mn2+ slightly more, stimulatory than Mg2+. Of the activities found in standard assay systems containing Mg2+, 58–78% (substrate UDP-glucose) and 0–38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg2+-dependent activities against the concentration of added Mg2+ were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.