bluuebunny/crossref_metadata_2025 huggingface dataset:
Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum
Authors: Stewart A C , Bendall D S
Published: August 2015

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.
Biochemical lesions in copper-deficient rats caused by secondary iron deficiency. Derangement of protein synthesis and impairment of energy metabolism
Authors: Weisenberg Emil , Halbreich Avraham , Mager Jacob
Published: August 2015

Severe copper deficiency was induced in rats by rearing nursing dams and their offsprings on a semisynthetic diet comprising all the requisite nutrients and trace metals except copper. The copper-deprived rats exhibited growth retardation, severe anaemia, loss of caeruloplasmin, decrease of cytochrome oxidase, accumulation of salt-soluble collagen and a drastic decrease in iron in plasma and liver. Apart from these characteristic signs of deficiency, a marked inhibition of protein synthesis was found to occur both in vivo and in cell-free liver preparations. The curtailed ability to carry out endogenously coded amino acid incorporation into protein contrasted with the unimpaired poly(U)-acid-directed phenylalanine polymerization. This inhibition pattern, as well as the attendant disaggregation of the liver polyribosomes, suggested that the primary biosynthetic lesion was located at the stage of peptide-chain initiation. Concurrently with this alteration there was a pronounced depletion of the hepatic ATP content, associated with a parallel depression of mitochondrial respiration and an enhancement of ATPase activity. Supplementation of the copper-deficient diet with a 2–4-fold excess of iron (relative to the standard diet) prevented growth retardation and anaemia and restored normal energy metabolism, as well as unimpaired protein-synthesizing capacity. The conclusion that these disturbances were primarily determined by the secondary iron deficiency was also borne out by the finding that similar alterations occurred in rats maintained on a copper-sufficient but iron-deficient diet. On the other hand, the iron-fortified diet failed to reverse the other signs of copper deficiency, namely the loss of caeruloplasmin, the diminished rate of cytochrome oxidase and the increase of soluble collagen. The interrelations between the various biochemical lesions induced by deprivation of copper or iron are discussed and the possible role of ATP depletion in determining the derangement of protein synthesis is considered.
Degradation of fructose-1,6-bisphosphate aldolase by cathepsin B
Authors: Bond J S , Barrett A J
Published: August 2015

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.
Circular dichroic, infrared and other studies on the protein component of pig brain thromboplastin
Authors: Howell R M , Rezvan H
Published: August 2015

1. A four-step procedure used to isolate the protein component (apoprotein III) of pig brain thromboplastin yielded approximately 25 mg from 500g of brain. 2. In the absence of detergent, apoprotein III had an apparent mol.wt. of 360 000 by gel-filtration, and, after electrophoresis on polyacrylamide gels in the presence of sodium docecyl sulphate, it appeared as a major protein band of mol.wt.59 000, suggesting the existence of polymeric and monomeric forms. 3. Chemical analyses of apoprotein III revealed that hydrophilic and hydrophobic amino acids were present in a ratio of 3:2, together with approx, 9% (w/w) of carbohydrate. 4. The far-u.v.c.d. and i.r. spectral data indicated that, like other membrane proteins, apoprotein III has a high percentage of unordered structure with lesser amounts of alpha and beta-forms. 5. Relipidation of apoprotein III to restore clotting activity caused no extensive alteration in the c.d. and i.r. spectra, indicating that the phospholipid associates with a comparatively small hydrophobic segment. The constrained unordered conformation, which makes the major contribution to the c.d. spectrum, probably forms a separate domain in the aqueous phase. The absence of any increase in the amplitude of both negative c.d. extrema, following relipidation, contrasted with the substantial increase observed in a helix-forming solvent and raises the possibility that the more stable polymeric form of apoprotein III is retained as the active form in the lipid phase. 6. We suggest that as a consequence of cell membrane damage, the recognition and activation of factor VII may involve minor changes of conformation that are dependent upon the flexibility inherent in an unordered secondary structure.
Methane production by the membranous fraction of Methanobacterium thermoautotrophicum
Authors: Sauer F D , Erfle J D , Mahadevan S
Published: August 2015

Intact membrane vesicles are required to synthesize methane from CO2 and H2 by disrupted preparations of Methanobacterium thermoautotrophicum cells. When membrane vesicles were removed by high-speed centrifugation at 226 600 g, the remaining supernatant fraction no longer synthesized methane. Alternatively, if vesicle structure was disrupted by passage through a Ribi cell fractionator at very high pressures (345 MPa), the bacterial cell extract, with all the particulate fraction in it, did not synthesize methane. Methyl-coenzyme M, a new coenzyme first described by McBride & Wolfe [(1971) Biochemistry 10, 2317–2324], was shown to stimulate methane production from CO2 and H2, as previously reported, but the methyl group of the coenzyme did not appear to be a precursor of methane in this reaction. No methyl-coenzyme M reductase activity was detected in the cytoplasmic fraction of M. thermoautotrophicum cells.
Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases
Authors: Cox R A , Kotecha S
Published: August 2015

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16–100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.
Effect of compound D-600 (methoxyverapamil) on gluconeogenesis and on acceleration of the process by α-adrenergic stimuli in rat kidney tubules
Authors: Saggerson E D , Carpenter C A
Published: August 2015

1. Tubule fragments were isolated from renal cortex of fed rats and glucose formation was measured after incubation with 5 mM-sodium lactate. 20 Compound D-600 (10-100 microM) decreased gluconeogenesis from lactate. This inhibition of the process by compound D-600 increased with increasing extracellular Ca2+ concentration, was overridden by noradrenaline and diminished by starvation for 24 h. 3. Inhibition of lactate-supported gluconeogenesis by compound D-600 was not prevented by the alpha 1-adrenoceptor antagonist thymoxamine. 4. Compound D-600 had little effect on gluconeogenesis from 2-oxoglutarate and increased gluconeogenesis from succinate. 5. Compound D-600 opposed stimulation of gluconeogenesis by noradrenaline or oxymetazoline (a selective alpha-adrenoceptor agonist) in a manner suggesting that compound D-600 is an alpha-adrenoceptor blocker. Oxymetazoline was more sensitive than noradrenaline to blockade by both compound D-600 and by the conventional alpha-adrenoceptor antagonist phentolamine. Noradrenaline became more sensitive to blockade by compound D-600 when extracellular Ca2+ was decreased. 6. Compound D-600 did not block stimulation of gluconeogenesis by angiotensin or cyclic AMP.
Restoration of guanine nucleotide- and fluoride-stimulated activity to an adenylate cyclase-deficient cell line with affinity-purified guanine nucleotide regulatory protein
Authors: Nielsen T B , Downs R W , Spiegel A M
Published: August 2015

A guanine nucleotide-binding protein purified from turkey erythrocytes by affinity chromatography confers both F— and guanine nucleotide-stimulation of adenylate cyclase to membranes from CYC- cells, a mutant cell line deficient in these responses. Interaction of turkey erythrocyte membranes with beta-adrenergic agonists before affinity chromatography, which is essential for binding of the guanine nucleotide regulatory protein to the affinity matrix, was also required for recovery of F—stimulation restoring activity in the affinity eluate.
Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings
Authors: Srivenugopal K S , Adiga P R
Published: August 2015

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.
A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei
Authors: Guarnieri C , Flamigni F , Caldarera C M
Published: August 2015

An alpha-tocopherol-binding macromolecule was isolated from the heart cytosol of rabbits fed for 1 month with an alpha-tocopherol-deficient diet. The amount of [3H]-tocopherol bound to nuclear chromatin was increased when the alpha-tocopherol-deficient heart nuclei were incubated in the presence of [3H]tocopherol-cytosol complex. In this condition, large amounts of [3H]tocopherol were associated with a subnuclear fraction that contained non-histone acidic proteins.
Relationship between acid-soluble carnitine and coenzyme A pools in vivo
Authors: Brass Eric P. , Hoppel Charles L.
Published: August 2015

The relationship between the acid-soluble carnitine and coenzyme A pools was studied in fed and 24-h-starved rats after carnitine administration. Carnitine given by intravenous injection at a dose of 60μmol/100g body wt. was integrated into the animal's endogenous carnitine pool. Large amounts of acylcarnitines appeared in the plasma and liver within 5min of carnitine injection. Differences in acid-soluble acylcarnitine concentrations were observed between fed and starved rats after injection and reflected the acylcarnitine/carnitine relationship seen in the endogenous carnitine pool of the two metabolic states. Thus, a larger acylcarnitine production was seen in starved animals and indicated a greater source of accessible acyl-CoA molecules. In addition to changes in the amount of acylcarnitines present, the specific acyl groups present also varied between groups of animals. Acetylcarnitine made up 37 and 53% of liver acid-soluble acylcarnitines in uninjected fed and starved animals respectively. At 5min after carnitine injection hepatic acid-soluble acylcarnitines were 41 and 73% in the form of acetylcarnitine in fed and starved rats respectively. Despite these large changes in carnitine and acylcarnitines, no changes were observed in plasma non-esterified fatty acid or β-hydroxybutyrate concentrations in either fed or starved rats. Additionally, measurement of acetyl-CoA, coenzyme A, total acid-soluble CoA and acid-insoluble CoA demonstrated that the hepatic CoA pool was resistant to carnitine-induced changes. This lack of change in the hepatic CoA pool or ketone-body production while acyl groups are shunted from acyl-CoA molecules to acylcarnitines suggests a low flux through the carnitine pool compared with the CoA pool. These results support the concept that the carnitine/acid-soluble acylcarnitine pool reflects changes in, rather than inducing changes in, the hepatic CoA/acyl-CoA pool.
Survey of virally mediated permeability changes
Authors: Foster K A , Gill K , Micklem K J , Pasternak C A
Published: August 2015

1. Sendai virus causes permeability changes when added to freshly isolated brain cells (cerebellum or ependymal cells) or to a culture of forebrain cells. 2. Sendai virus causes permeability changes when added to organ cultures of ferret lung or nasal turbinate. Influenza virus causes no permeability changes under these conditions. 3. Rabies virus and vesicular-stomatitis virus, in contrast with Sendai virus, do not cause permeability changes in BHK cells or Lettrée cells. 4. Serum from patients suffering from viral hepatitis does not cause permeability changes in human leucocytes; addition to Sendai virus causes permeability changes. 5. It is concluded that permeability changes accompanying viral entry occur only with certain types of paramyxovirus, but that there is little restriction on cell type. 6. MDBK cells infected with Sendai virus show permeability changes during viral release, similar to those that occur during viral entry. Because these changes do not appear to be restricted to paramyxoviruses, they may have considerable clinical significance.
The effect of calmodulin on the active calcium-ion transport and (Ca2+ + Mg2+)-dependent ATPase in microsomal fractions of smooth muscle compared with that in erythrocytes and cardiac muscle
Authors: Wuytack F , De Schutter G , Casteels R
Published: August 2015

The Ca2+ uptake and the (Ca2+ + Mg2+)-dependent ATPase of the porcine coronary-artery smooth-muscle microsomal fraction (‘microsomes’) are only slightly stimulated by calmodulin. The Ca2+ uptake after 2 min in the absence of oxalate, corrected for the ATP-independent binding, increased by a factor of 1.44, whereas the (Ca2+ + Mg2+)-dependent ATPase is stimulated 1.39 times. These findings contrast with the effect observed in human erythrocyte ‘inside-out’ microsomes. In these vesicles calmodulin increases the Ca2+ uptake after 20 min in an oxalate-free medium and the (Ca2+ + Mg2+)-dependent ATPase respectively by a factor of 3.82 and 6.18. The magnitude of the calmodulin stimulation of the Ca2+ transport in coronary-artery microsomes is similar to that observed in heart microsomes.
Cellular uptake of retinoic acid in vitro
Authors: Banerjee C K , Sani B P
Published: August 2015

Incubation of chick embryo skin and mouse colon tumour 26 with [3H]retinoic acid resulted in the formation of a complex of retinoic acid and its cellular binding protein both in cytosol and in nuclei. Formation of the ligand—protein complex was temperature-dependent and increased with increases in retinoic acid concentration in the incubation medium. About 3—8% of the ligand present in the cytosol was associated with the nuclei.
Purification, properties and synthesis of δ-aminolaevulinate dehydratase from Neurospora crassa
Authors: Chandrika S R , Padmanaban G
Published: August 2015

Delta-aminolaevulinate dehydratase, the second and rate-limiting enzyme of the haem-biosynthetic pathway, was purified 300-fold from induced cultures of Neurospora crassa. The native enzyme has a mol.wt. of about 350000, whereas the salt-treated enzyme after incubation at 37 degrees C for 10 min has a mol.wt. of about 232000. The mol.wt. of the subunit is about 38000. Antibodies to the purified enzyme were raised in rabbits. By using radiolabelling and immunoprecipitation techniques it was shown that addition of iron and laevulinate to iron-deficient cultures brings about a significant increase in the synthesis of the enzyme, and protoporphyrin, the penultimate end product of the pathway, represses enzyme synthesis.
Further studies on histamine release from rat mast cells in vitro induced by peptides. Characteristics of a synthetic intermediate with potent releasing activity
Authors: Roy P D , Moran D M , Bryant V , Stevenson R , Stanworth D R
Published: August 2015

Previous studies on histamine release by corticotropin peptides and melittin peptides were extended, leading to the identification of a synthetic peptide intermediate, Lys(Z)-Arg(NO2)-Arg(NO2)OMe, (I) as an active non-cytolytic histamine releaser from rat mast cells. However, significant differences in the releasing capacity of optical isomers of this compound, and of Lys-Lys-Arg-ArgOMe [methyl ester of corticotropin-(15-18)-tetrapeptide; ‘basic core’] were observed, with the L-forms being markedly more active. A study of various analogues of the tripeptide compound (I) indicated that the structural basis for mast-cell triggering by such peptidic agents was highly specific. The relevance of these observations to the immunologically induced histamine-release processes is discussed.
Ligand-binding properties of proalbumin Christchurch
Authors: Reed R G , Peters T , Brennan S O , Carrell R W
Published: August 2015

Proalbumin Christchurch, a circulating variant of human serum albumin, is secreted from the liver without cleavage of the hexapeptide situated at the N-terminal end of the peptide chain of proalbumin. We compared ligand-binding properties of proalbumin Christchurch and of normal albumin A from the same individual in order to test the effect of the presence of the hexapeptide. The two albumin forms exhibited similar affinities for palmitate, bilirubin, 8-anilinonaphthalene-1-sulphonate and Bromocresol Green. The patterns of endogenous fatty acids bound to the two forms of albumin were slightly different, although the differences were probably not of physiological significance. From these studies it would appear that the propeptide of proalbumin does not alter the protein conformation in such a way as to alter binding sites for organic anions.
New proline-rich proteins in isolated insect Z-discs
Authors: Sainsbury G M , Bullard B
Published: August 2015

Z-discs were isolated from Lethocerus (waterbug) flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. Sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis confirmed a previous report that major Z-disc proteins had subunit mol.wts of 200 000, 180 000, 105 000, 95 000, 42 000 and 35 000. A protein of subunit mol.wt 25 000 was found in once-washed Z-discs but was degraded or was removed by successive washes. In addition, a protein of high molecular weight (less than 300 000) was found in Z-discs. Proteins of subunit mol.wts. 42 000, 35 000 and 25 000 were individually sliced from SDS/polyacrylamide gels and eluted. Amino acid analysis showed that the 35 000-subunit-mol.wt. protein was not, as was previously suggested, tropomyosin, but was a distinct Z-disc protein rich in proline. Calculations based on the amino acid analysis showed that this protein contained substantial hydrophobic regions. Preliminary investigations into the isoelectric point and a method of isolation of the 35 000-subunit-mol.wt. Z-disc protein are described. This protein was found in slices cut from SDS/polyacrylamide-gel electrophoretograms of whole myofibrils. The protein of 42 000 subunit mol.wt. was shown by amino acid analysis to be actin and the 25 000-subunit-mol.wt. Z-disc protein was proline-rich.
A comparison of potato and vertebrate lactate dehydrogenases
Authors: Poerio E , Davies D D
Published: August 2015

A 2000-fold purification of L(+)-lactate dehydrogenase from potatoes is reported. Five isoenzymes of lactate dehydrogenase can be detected in crude extracts of potato, and three of these are present in the purified preparation. The enzyme (mol.wt. 150 000), which is composed of four subunits (mol.wt. 37 500), is active with the same oxo acids and hydroxy acids that have been reported as substrates with the same oxo acids and hydroxy acids that have been reported as substrates for vertebrate lactate dehydrogenases. These similarities between potato and vertebrate lactate dehydrogenases contrast sharply with some other reports on potato lactate dehydrogenase. These discrepancies are discussed in relation to the proposition that vertebrate and potato lactate dehydrogenases share a common evolutionary origin.
Magnetization curves of haemoproteins measured by low-temperature magnetic-circular-dichroism spectroscopy
Authors: Thomson A J , Johnson M K
Published: August 2015

The magnetic-circular-dichroism (m.c.d.) spectra of methymyoglobin cyanide and oxidized horse heart cytochrome c were measured in the region of the Soret band over a range of temperatures from 1.5 to 50 K and in fields from 0 to 5T. A similar study has been made with reduced bovine heart cytochrome c oxidase, which contains one high-spin ferrous haem, namely a3. M.c.d. magnetization curves characteristic of an isolated Kramer's ground state with spin S = 1/2. These curves contrast with the magnetization curve of the high-spin ferrous haem with spin S = 2. The electronic ground state of the latter compound contains zero-field components that are thermally accessible over the temperature range of the experiment. Hence the magnetization curves are a complex nested set. The magnetization curves of the S = 1/2 proteins were analysed and it is shown that it is possible to make estimates of the ground-state g-factors even in the presence of rhombic anisotropy, provided that some knowledge of the polarizations of the electronic transitions is available. The striking difference between the m.c.d. magnetization curves of a simple S = 1/2 paramagnet and magnetically complex ground state should prove extremely useful when m.c.d. spectroscopy is sued to probe the magentic properties of metal centres in proteins, and should have wide application beyond the field of haemoproteins.
Human cathepsin H
Authors: Schwartz W N , Barrett A J
Published: August 2015

Cathepsin H was purified from human liver by a method involving autolysis and acetone fractionation, and chromatography on DEAE-cellulose, Ultrogel AcA 54, hydroxyapatite and concanavalin A-Sepharose. The procedure allowed for the simultaneous isolation of cathepsin B and cathepsin D. Cathepsin H was shown to consist of a single polypeptide chain of 28 000 mol.wt., and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. The enzyme existed in multiple isoelectric forms, the two major forms having pI values of 6.0 and 6.4; it hydrolysed azocasein (pH optimum 5.5), benzoylarginine 2-naphthylamide (Ba-Arg-NNap), leucyl 2-naphthylamide (Arg-NNap), (pH optimum 6.8). Arg-NNap and Arg-NMec, unlike Bz-Arg-NNap-, were not hydrolysed by human cathepsin B. Cathepsin H was similar to cathepsin B in being irreversibly inactivated by exposure to alkaline pH. Sensitivity to chemical inhibitors by 1 microM-leupeptin, which gave essentially complete inhibition of the other lysosomal cysteine proteinases, cathepsins B and L.
Stereospecific distribution of plamitic acid in the triacylglycerols of rat adipocytes. Effects of varying the composition of the substrate fatty acid in vitro
Authors: Christie William W. , Hunter Margaret L.
Published: August 2015

The effects of inclusion of different fatty acids in the medium on the rate of esterification of palmitic acid and its stereospecific distribution among the three positions of the triacyl-sn-glycerols by preparations of rat adipocytes in vitro have been determined. Myristic acid, stearic acid, oleic acid and linoleic acid were used as diluents and the concentration of the combined unesterified fatty acids in the medium was held constant; only the proportion of palmitic acid was varied. The amount of palmitic acid esterified was always linearly related to its relative concentration in the medium and was not significantly affected by the nature of the diluent fatty acid chosen. Constant relative proportions were recovered in triacylglycerols and in intermediates in each instance. The amount of palmitic acid esterified to each of the positions of the triacyl-sn-glycerols was linearly dependent on the relative proportion in the medium but the nature of the relationship was markedly influenced by which fatty acid was present. When stearic acid was present, simple relationships were found over the whole range tested. When either myristic acid, oleic acid or linoleic acid was present, abrupt changes in the manner of esterification of palmitic acid were observed in position sn-1 when the relative concentrations of palmitic acid and the diluent reached critical values, which differed with each fatty acid. In position sn-2 when oleic acid or linoleic acid was present, a similar change was observed, and in position sn-3 it was obtained with myristic acid as diluent. The results are discussed in terms of changes in the relative affinities of the acyltransferases for palmitic acid. Palmitic acid was esterified into various molecular species in proportions that indicated acylation with non-correlative specificity at higher relative concentrations but not at lower.
Use of fluorescent probes in the study of phospholipid–sterol bilayers
Authors: Wharton S A , De Martinez S G , Green C
Published: August 2015

1. The transfer of excitation energy between the fluorescent probes 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyl)stearic acid and the cholesterol analogue cholesta-4,6-dien-3-one in phosphatidylcholine liposomes has been investigated. 2. The results indicate that probes and steroid are randomly distributed in the bilayer at steroid concentrations up to 35 mol%. 3. The degree of polarization of diphenylhexatriene fluorescence increases with increasing cholesterol content. Other sterols, differing in structure in the region of the polar group or in the side chain at position-17, produce similar but not identical effects. 4. the results are consistent with the proposal that diphenylhexatriene gives a general picture of the state of the bilayer and that there is no segregation of sterols in liquid-crystalline phosphatidylcholine bilayers.
The effect of hyperphenylalaninaemia on glycine metabolism in developing rat brain
Authors: Isaacs C E , Greengard O
Published: August 2015

The brains of 3–16-day-old rats that were rendered hyperphenylalaninaemic by daily injections of alpha-methylphenylalanine plus phenylalanine were subjected to biochemical analysis. Fluctuations throughout the treatment period in the concentrations of branched-chain amino acids, methionine and serotonin were in agreement with the known interference of excess plasma phenylalanine with transport. The glycine content, however, became abnormal only by day 5, remained so through the treatment, and the elevation was equally apparent at 4, 8 or 24 h after the last daily injections. On the last day of treatment there were small increases in the taurine, glutamate, aspartate and 4-aminobutyrate concentrations, attributable mainly to the diencephalon or brain stem. After day 3 of treatment there were persistent elevations in the specific activity of phosphoserine phosphatase and glycine synthase (but not serine hydroxymethyltransferase) of the brain in each of the regions analysed. The observations indicate that chronic hyperphenylalaninaemia interferes with the normal regulation of intracerebral glycine metabolism during a critical period of early postnatal development, and suggest that the resulting excess in this amino acid (particularly marked in the cortex) contributes to the behavioural abnormalities that these animals exhibit in later life.
Uptake of safranine by cardiac mitochondria. Competition with calcium ions and dependence on anions
Authors: Harris E J , Baum H
Published: August 2015

Some characteristics of the energized uptake of safranine by rat heart mitochondria were studied. When monitored by changes in differential absorbance (between 524 and 554 nm) of a whole suspension in safranine-containing medium, the changes seen are not linearly related to the quantity of the safranine moving. It happens coincidentally that the changes observed are nearly linearly related to the logarithm of the ratio between the accumulated safranine and its residual concentration in the medium; this explains why the changes of absorbance have been found by other authors to be linearly related to the logarithms of the ratio of internal/external concentrations of such other cations as are permeable. The uptake process appears to compete for energy with Ca2+ uptake and vice versa. Energized safranine uptake has an anion requirement, which is seen when movement of endogenous Pi has been inhibited; the small residual safranine uptake obtained when energy is provided in the presence of mersalyl may be attributable to internal Pi. However, a limited anion-independent energized uptake of safranine, in exchange for internal K+, may be elicited in the presence of nigericin. Adding ATP to the energized system in the presence of an inhibitor of Pi movement elicits an additional uptake of safranine that is oligomycin-sensitive and that probably arises on account of generation of internal Pi by hydrolysis of the entering ATP.
Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo
Authors: Munniksma J , Noteborn M , Kooistra T , Stienstra S , Bouma J M W , Gruber M , Brouwer A , Praaning-van Dalen D , Knook D L
Published: August 2015

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.
The interrelation of the two c-type cytochromes in Rhodopseudomonas sphaeroides photosynthesis
Authors: Wood P M
Published: August 2015

Photosynthetic electron flow in the bacterium Rhodopseudomonas sphaeroides involves two c-type cytochromes, one membrane-bound and the other a soluble protein, cytochrome c2. Membranes deficient in cytochrome c2 were used for photo-oxidation studies, with and without the addition of purified cytochrome c2. The results favour a series interrelation, membrane cytochrome c-cytochrome c2-reaction centre.
Thyroid-hormone effects on ornithine decarboxylase
Authors: Chideckel E W , Rozovski S J , Belur E R
Published: August 2015

We examined thyroidectomized, normal and hyperthyroid rats and found that ornithine decarboxylase activity was directly correlated with thyroid functional state in heart and liver and unaffected in brain, testes and spleen, phenomena that correlate with the known effect of thyroid hormone on protein synthesis.
Uptake of phospholipid-depleted chylomicrons by the perfused rat liver
Authors: Borensztajn J , Kotlar T J , McNeill B J
Published: August 2015

1. Rat lymph chylomicrons were depleted of their surface phospholipids by treatment with pure phospholipase A2 from Crotalus adamanteus venom. 2. About 80% of the phospholipids could be removed from the chylomicrons without any apparent effect on their size, neutral lipid composition or qualitative profile of their tetramethylurea-soluble apoproteins. 3. Phospholipid-depleted chylomicrons were rapidly taken up whole by liver cells when perfused through isolated rat liver preparations. The rate of uptake was dependent on the extent of phospholipid depletion and reached a maximum (4-6.5-fold greater than control chylomicrons) when 80% of the phospholipids had been removed. 4. It is speculated that the hepatic uptake of phospholipid-depleted chylomicrons occurs by a mechanism to that of chylomicron-remnants uptake.
Increased transcription and decreased degradation control and recovery of liver ribosomes after a period of protein starvation
Authors: Conde R D , Franze-Fernández M T
Published: August 2015

In the livers of 5-days-protein-depleted mice there is a decrease of 47% of the ribosome mass. When these animals are fed with an adequate diet, ribosome content is restored to the normal value after 1 day of re-feeding. The mechanisms underlying this phenomenon were studied. It was found that: (1) the activity of RNA polymerase I in the nuclei of livers from re-fed animals showed an enhancement of about 2-fold compared with the activity in normal and protein-depleted liver nuclei; (2) ribosome degradation, measured by the disappearance of radioactivity from ribosomal proteins previously labelled by the administration of NaH14CO3 to the mice, stopped during the first day after re-feeding.
Comparative studies on amniotic fluid and plasma fibronectins
Authors: Ruoslahti E , Engvall E , Hayman E G , Spiro R G
Published: August 2015

Human fibronectin was isolated from second-trimester amniotic fluid, from amniotic fluid obtained at term and from adult plasma. The amniotic-fluid fibronectins had a slightly higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than the plasma fibronectin. Early- and late-amniotic-fluid fibronectin had 9.5 and 9.6% carbohydrate respectively, whereas plasma fibronectin had 5.8%. The amniotic-fluid fibronectins had similar mannose and sialic acid contents to plasma fibronectin, but greater amounts of glucosamine, galactosamine, galactose and fucose. There were no detectable differences in the amino-acid composition of amniotic-fluid and plasma fibronectins, and the patterns of peptides obtained after tryptic digestion of fibronectin from the two sources showed extensive similarities. Fibronectins from plasma and amniotic fluid were equally active in promoting cell attachment and were immunologically indistinguishable. These results show that fibronectin from amniotic fluid is more heavily glycosylated than plasma fibronectin or previously analysed fibronectins from cultured fibroblasts. The observed differences in glycosylation may be related to cell type and/or stage of development.
Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver
Authors: Kajander E O , Raina A M
Published: August 2015

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.
Purification and characterization of subcomponent C1q of the first component of mouse complement
Authors: Yonemasu K , Sasaki T
Published: August 2015

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 × 10(13)-4 × 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.
Concanavalin A, a receptor protein with apparently co-operative binding characteristics
Authors: Köttgen E , Bauer C , Ehlerding U , Gerok W
Published: August 2015

Laser nephelometry is a suitable technique for the quantitative determination and differentiation of both lectins and glycoconjugates in the low-picomolar range. Simultaneously this method renders possible investigations on the specificity and mode of interaction between lectins and different ligands. The results demonstrate that the degree of co-operativity between concanavalin A and the respective glycoconjugate is dependent on the presence of hydrophobic binding sites and can be substantially altered by conformational changes of the ligand. The transition from apotransferrin to Fe3+-transferrin induces a transformation of the sigmoidal-shaped binding curve to a hyperbolic one. Hence, at low concentrations, Fe3+-transferrin is bound far better than apotransferrin, whereas maximal binding is nearly identical. After removal of N-acetylneuraminate, concanavalin A is less efficient in differentiating between the Fe3+-charged and Fe3+-free (apo) forms of transferrin.
Nature of the thiamin-binding protein from chicken egg yolk
Authors: Muniyappa K , Adiga P R
Published: August 2015

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate–AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.
The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro
Authors: Baydoun E A H , Northcote D H
Published: August 2015

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.
Degradation of keratan sulphate by β-N-acetylhexosaminidases A and B
Authors: Ludolph T , Paschke E , Glössl J , Kresse H
Published: August 2015

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay–Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH–activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.
Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition
Authors: Heldin C H , Westermark B , Wasteson A
Published: August 2015

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 × 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.