bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis
Authors: Baici A , Seemüller U
Published: August 2015

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 × 10(6)M-1 X S-1 and 8 × 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 × 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.
Is N-acetylglutamate a short-term regulator of urea synthesis?
Authors: Lund P , Wiggins D
Published: August 2015

A method is described for determining N-acetylglutamate as glutamate. N-Acetylglutamate content of hepatocytes from 48 h-starved rats is high. It shows no parallelism with rates of urea synthesis from glutamine. We question its accepted function as a short-term regulator of urea synthesis.
Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure
Authors: Schiaffonati L , Bardella L , Cairo G , Giancotti V , Bernelli-Zazzera A
Published: August 2015

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.
Bradykinin stimulates bone resorption and lysosomal-enzyme release in cultured mouse calvaria
Authors: Gustafson G T , Lerner U
Published: August 2015

The effect of bradykinin on bone resorption was studied in cultures of newborn-mouse calvaria. Bradykinin (0.03 microM, 1 microM) stimulated the release of 45Ca2+ from bones dissected out from mice prelabelled in vivo with 45Ca. Bradykinin (1 microM) also augmented the release of stable calcium (40Ca), Pi and the lysosomal enzyme beta-glucuronidase. The stimulatory effect of bradykinin on mineral mobilization and lysosmal -enzyme release could be blocked by indomethacin. It is speculated that concomitant generation of thrombin and bradykinin in areas of trauma and inflammation may induce resorption of nearby bone tissue.
Exposure of thyroxine residues in human thyroglobulin. Two-site binding studies
Authors: Byfield P G H , Clingan D , Himsworth R L
Published: August 2015

Human thyroglobulin (Tg) could be adsorbed through one of its thyroxine (T4) residues by either of two T4-binding antibodies which had been covalently attached to Sepharose- CL4B . The antibodies used were (i) a purified human autoantibody specific for a T4-containing epitope in human Tg, or (ii) a rabbit antibody raised against T4 conjugated to bovine albumin side chains. Tg adsorbed by either immobilized antibody could then itself adsorb either type of antibody free in solution on to a further T4 residue. At least two T4 residues in human Tg are therefore sufficiently exposed to interact with T4-binding antibodies. Furthermore, these T4 residues are sufficiently far apart to allow the binding of two immunoglobulin molecules simultaneously. Previous observations of a marked preference by human autoantibodies for one of the T4-containing epitopes in Tg therefore reflect a higher binding energy with that epitope rather than an inability to interact with others. The T4-containing epitope which preferentially reacts with human Tg autoantibodies must therefore have a distinctive topography.
The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects
Authors: Patel D D , Pullinger C R , Knight B L
Published: August 2015

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.
Fast-atom-bombardment mass spectrometry and electron-impact direct-probe mass spectrometry in the structural analysis of oligosaccharides with special reference to the poly(N-acetyl-lactosamine) series
Authors: Hounsell E F , Madigan M J , Lawson A M
Published: August 2015

A comparison has been made of positive- and negative-ion fast-atom-bombardment (FAB) and electron-impact (EI) mass spectrometry for analysis of oligosaccharides and alditols containing alternating and consecutive sequences of neutral and acetamido sugars. Among these were novel chemically synthesized tetrasaccharides with Ii antigen activities. FAB ionization has the advantage that it is applicable to non-derivatized oligosaccharides and it can determine Mr. However, the abundance of fragment ions providing structural information and the amount of material required for analysis (1-50 nmol) varied from sample to sample. In contrast, EI mass spectrometry of 5 nmol of permethylated or peracetylated oligosaccharides reliably gives all the fragment ions formed by cleavage across the glycosidic bonds.
Kinetic mechanism and specificity of bovine milk sulphydryl oxidase
Authors: Sliwkowski M X , Swaisgood H E , Clare D A , Horton H R
Published: August 2015

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.
Repair of depurinated DNA with enzymes from rat liver chromatin
Authors: Goffin C , Verly W G
Published: August 2015

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5′----3′-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5′ to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.
Inhibition of human liver cathepsin L by α2 cysteine-proteinase inhibitor and the low-Mr cysteine proteinase inhibitor from human serum
Authors: Pagano M , Esnard F , Engler R , Gauthier F
Published: August 2015

The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.
Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells
Authors: Van Schravendijk C F H , Hooghe-Peters E L , De Meyts P , Pipeleers D G
Published: August 2015

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.
Age-related decrease in the link-stability of proteoglycan aggregates formed by articular chondrocytes
Authors: Plaas A H K , Sandy J D
Published: August 2015

Chondrocytes were isolated from the articular cartilage of rabbits aged between 6 and 50 weeks and labelled with [35S]sulphate after 48 h in monolayer culture. The percentage of the total proteoglycan monomers synthesized by each culture that were present as link-stabilized aggregates was shown to be about 83% at 6, 9 and 12 weeks, 73% at 15 weeks, 48% at 30 weeks and 32% at 50 weeks. The proliferative activity of the cells in culture also decreased markedly with the age of the donor. The results suggest that aging of chondrocytes in vivo is accompanied by a decrease in their capacity for link-protein synthesis.
Triacylglycerol synthesis in goat mammary gland. Factors influencing the esterification of fatty acids synthesized de novo
Authors: Hansen H O , Grunnet I , Knudsen J
Published: August 2015

ATP alone had no effect on incorporation of fatty acids synthesized de novo and membrane-bound diacylglycerol into triacylglycerol. Combined addition of ATP and Mg2+ totally inhibits incorporation of fatty acids synthesized de novo and stimulated incorporation of membrane-bound diacylglycerol. ATP, Mg2+ and glycerol 3-phosphate stimulate incorporation of fatty acids synthesized de novo into triacylglycerol, but inhibited the incorporation of membrane-bound diacylglycerol. Diacylglycerol generated in situ was shown to be superior to diacylglycerols preloaded on the membrane as substrate for the diacylglycerol acyltransferase. A model is proposed to explain the effect of absorbed exogenous fatty acid on fatty acid synthesis de novo in goat mammary gland.
A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion
Authors: Liew C C , Halikowski M J , Zhao M S
Published: August 2015

[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.
Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein
Authors: Powers J C , McRae B J , Tanaka T , Cho K , Cook R R
Published: August 2015

A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold ‘better’ than those in the glycine series. The S3 subsite was found to ‘prefer’ lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.
The effects of colchicine on secretion into bile of bile salts, phospholipids, cholesterol and plasma membrane enzymes: bile salts are secreted unaccompanied by phospholipids and cholesterol
Authors: Barnwell S G , Lowe P J , Coleman R
Published: August 2015

Colchicine, a drug which interferes with microtubular function, has no effect on the secretion of taurodehydrocholate into bile; it is therefore suggested that bile salts are unlikely to be packaged in vesicles during cellular transit from sinusoidal to canalicular membranes. Colchicine greatly reduces the secretion of phospholipid and cholesterol into bile; it is suggested that this is due to an interruption in the supply of vesicles bringing lipids to repair the canalicular membrane during bile salt output. In the absence of the protective effect of a continuous supply of repair vesicles, micelleforming bile salts damage the canalicular membrane; the increased concentration of plasma membrane enzymes in bile and the increased aspartate aminotransferase activity in plasma and bile are evidence of this damage. Damage to the canalicular membrane may also be an explanation for the reduction in taurocholate transport and the taurocholate-induced cholestasis which are seen with colchicine-treated livers. Such membrane damage is not observed in colchicine-treated livers during the secretion of the non-micelle forming bile salt, taurodehydrocholate.
The effects of magnesium ions on the interactions of ox brain and liver glutamate dehydrogenase with ATP and GTP
Authors: McCarthy A D , Tipton K F
Published: August 2015

The effects of ATP and GTP on the activities of ox liver and brain glutamate dehydrogenase were determined in the absence and presence of added Mg2+ ions. Although GTP was an inhibitor of the enzyme reaction assayed in the direction of NAD+ reduction, the magnesium complex of this nucleotide had no effect on the activity. Similarly the magnesium complex of ATP was without effect on the activity of the enzyme although the free nucleotide was an activator. These results suggest that it is important to take account of magnesium complex formation when considering the regulatory actions of these nucleotides.
Increased activity of plasminogen activators during involution of the rat ventral prostate
Authors: Rennie P S , Bouffard R , Bruchovsky N , Cheng H
Published: August 2015

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.
Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility
Authors: Cole S C J , Yon R J
Published: August 2015

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.
Glucose-dependent induction of acetyl-CoA carboxylase in rat hepatocyte cultures
Authors: Giffhorn S , Katz N R
Published: August 2015

The carbohydrate-dependent long-term regulation of acetyl-CoA carboxylase was studied in primary hepatocyte cultures from adult rats. (1) The enzyme activity was increased 2-fold either by elevation of the glucose concentration to 20mM or by enhancement of the insulin concentration to 0.1 microM. Simultaneous increases in glucose and insulin resulted in a 5-fold increase in the enzyme activity. (2) As shown by immunochemical titration, the enhancement of the enzyme activity was due to an increase in the enzyme protein. (3) Incorporation of [35S]methionine and immunoprecipitation of the enzyme revealed that the increase in enzyme protein was due to an increased rate of enzyme synthesis. The rate of enzyme degradation remained essentially unchanged. (4) The glucose- and insulin-dependent induction of acetyl-CoA carboxylase was prevented by the addition of alpha-amanitin (10 microM) or cordycepin (10 microM), indicating a transcriptional regulation of the enzyme level. (5) The parallel induction of acetyl-CoA carboxylase and of ATP citrate lyase indicates a co-ordinate long-term regulation of lipogenic enzymes.
Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351
Authors: Khoo T C , Cowan D A , Daniel R M , Morgan H W
Published: August 2015

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8×10(4)M-1 (for the low-affinity sites) and 7.5×10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.
Direct structural information for the copper site of dopamine β-mono-oxygenase obtained by using extended X-ray-absorption fine structure
Authors: Hasnain S S , Diakun G P , Knowles P F , Binsted N , Garner C D , Blackburn N J
Published: August 2015

Copper K-edge e.x.a.f.s (extended X-ray-absorption fine structure) was measured for dopamine beta-mono-oxygenase in aqueous solution. Comparison with the Cu K-edge e.x.a.f.s. of bovine erythrocyte superoxide dismutase shows a close resemblance. Detailed analysis of the e.x.a.f.s. indicates that the copper atom is bound to four imidazole groups at 0.201 nm with one or two oxygen atoms at 0.23 nm.
Variation in quantity and extractability of the 148-kilodalton cartilage protein with age
Authors: Paulsson M , Inerot S , Heinegård D
Published: August 2015

The occurrence of non-collagenous matrix proteins was studied in samples of tracheal cartilage from steers of different ages. The amounts of the 148 kDa cartilage protein in 4M-guanidinium chloride extracts and in subsequent trypsin digests of the extraction residues were determined by radioimmunoassay. Surprisingly, the 148 kDa-protein antigenicity was not changed by tryptic digestion, even though the protein was extensively degraded. The amount of the 148 kDa protein increased dramatically with age, both in the guanidinium chloride extract and in the subsequent tryptic digest, and reached maximal values at about 3 and 8 years respectively. The increase of the guanidinium chloride-soluble pool preceded that of the trypsin-digestible pool, possibly indicating a metabolic relationship. The ratio between the trypsin-digestible and guanidinium chloride-soluble pools increased continuously, and at 12 years of age close to 90% of the total 148 kDa protein detected was insoluble in guanidinium chloride. At all ages, the bulk of the cartilage collagen was insoluble both to extraction with guanidinium chloride and to tryptic digestion. The decreasing extractability of the 148 kDa protein was therefore not secondary to changes in the solubility of the collagen network. Other cartilage proteins, such as the link proteins and the 36 kDa protein, showed much smaller quantitative variations of a different character.
The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage
Authors: Roughley P J , White R J , Poole A R , Mort J S
Published: August 2015

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.
An investigation into the solution structures of two self-complementary DNA oligomers, 5′-d(C-G-T-A-C-G) and 5′-d(A-C-G-C-G-C-G-T), by means of nuclear-Overhauser-enhancement measurements
Authors: Gronenborn A M , Clore G M , Kimber B J
Published: August 2015

A 500 MHz 1H-n.m.r. study on two self-complementary alternating pyrimidine-purine oligodeoxyribonucleotides, 5′-d(C-G-T-A-C-G) and 5′-d(A-C-G-C-G-C-G-T), is presented. By using the proton-proton nuclear Overhauser effect virtually complete assignments are obtained and a large number of interproton distances [113 in the case of 5′-d(C-G-T-A-C-G) and 79 in the case of 5′-d(A-C-G-C-G-C-G-T)], both intra- and inter-nucleotide, are determined. The interproton-distance data are consistent with an overall right-handed B-DNA-type structure for both oligonucleotides, in agreement with their B-type c.d. spectra. However, whereas 5′-d(C-G-T-A-C-G) adopts a conventional B-type structure with a mononucleotide repeating unit, the interproton-distance data provide evidence that 5′-d(A-C-G-C-G-C-G-T) has a dinucleotide repeating unit consisting of alternation in glycosidic bond and sugar pucker conformations.
Rapid accumulation of inositol phosphates in isolated rat superior cervical sympathetic ganglia exposed to V1-vasopressin and muscarinic cholinergic stimuli
Authors: Bone E A , Fretten P , Palmer S , Kirk C J , Michell R H
Published: August 2015

An accumulation of 3H-labelled inositol phosphates is observed when prelabelled rat superior cervical sympathetic ganglia are exposed to [8–arginine]vasopressin or to muscarinic cholinergic stimuli. The response to vasopressin is much greater than the response to cholinergic stimuli. The response to vasopressin is blocked by a V1-vasopressin antagonist, and oxytocin is a much less potent agonist than vasopressin. Vasopressin causes no increase in the cyclic AMP content of ganglia. These ganglia therefore appear to have functional V1-vasopressin receptors that are capable of activating inositol lipid breakdown, but no V2-receptors coupled to adenylate cyclase. The first [3H]inositol-labelled products to accumulate in stimulated ganglia are inositol trisphosphate and inositol bisphosphate, suggesting that the initiating reaction in stimulated inositol lipid metabolism is a phosphodiesterase-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate (and possibly also phosphatidylinositol 4-phosphate). This response to exogenous vasopressin occurs in ganglia incubated in media of reduced Ca2+ concentration. The physiological functions of the V1-vasopressin receptors of these ganglia remain unknown.
Effects of an adenosine-receptor antagonist on insulin-resistance in soleus muscle from obese Zucker rats
Authors: Challis R A , Budohoski L , McManus B , Newsholme E A
Published: August 2015

The decreased sensitivity of glycolysis to insulin seen in isolated soleus muscles from genetically obese Zucker rats was abolished by addition of the adenosine-receptor antagonist 8-phenyltheophylline to the incubation medium; 8-phenyltheophylline had no effect on the sensitivity of glycogen synthesis to insulin. These findings suggest that changes in the sensitivity of glucose utilization by muscles of genetically obese rats may be explained, in part, by a modification in either the concentration of adenosine or the affinity of adenosine receptors in skeletal muscle.
Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma
Authors: Müllertz S , Thorsen S , Sottrup-Jensen L
Published: August 2015

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.
Purification and some properties of the d-lactate-2-sulphatase of Pseudomonas syringae GG.
Authors: Crescenzi A M V , Dodgson K S , White G F
Published: August 2015

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.
Nucleotide sequence encoding the iron-sulphur protein subunit of the succinate dehydrogenase of Escherichia coli
Authors: Darlison M G , Guest J R
Published: August 2015

The nucleotide sequence of a 961 base-pair segment of DNA containing the sdhB gene, which encodes the iron-sulphur protein subunit of the E. coli succinate dehydrogenase, has been determined. The sdhB structural gene comprises 711 base pairs (237 codons, excluding the translational initiator and terminator). It is separated by a 15 base-pair intergenic region from the preceding flavoprotein gene (sdhA) and is the distal gene of an operon that also includes genes (sdhC and D) encoding two hydrophobic subunits, sdhCDAB. The distal end of the sdh operon is linked to the 2-oxoglutarate dehydrogenase gene (sucA) by a complex region of dyad symmetry that is homologous with several potential intercistronic regulatory elements or transcriptional attenuators. The sdhB structural gene encodes a polypeptide of Mr26637 that is strikingly homologous with the iron-sulphur protein subunit of fumarate reductase (38% identity, increasing to 58% when conservative changes are included). Both subunits contain 11 cysteine residues, 10 being conserved in three clusters resembling those found in ferredoxins. This work completes the sequence of a 9897 base-pair segment of DNA containing seven tricarboxylic acid cycle genes encoding three enzymes or enzyme complexes, citrate synthase (gltA), succinate dehydrogenase (sdh), and the 2-oxoglutarate dehydrogenase complex (suc), that are organized thus: gltA-sdhCDAB-sucAB.
The inactive MoFe protein (NifB−Kp1) of the nitrogenase from nif B mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed
Authors: Hawkes T R , Smith B E
Published: August 2015

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.
Pulsed cytochrome c oxidase from the thermophilic bacterium PS3
Authors: Sone N , Naqui A , Kumar C , Chance B
Published: August 2015

A caa3-type terminal cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 containing three subunits showed conversion from resting into pulsed form. Upon pulsing (reduction and re-oxidation), the cytochrome c oxidase activity increased over 10-fold. This enhanced activity of the pulsed enzyme gradually decayed. Addition of phospholipids, necessary for the enzyme activity, did not affect this decay process. Small changes in the absorption spectrum were observed for the resting-into-pulsed transition and for H2O2 ligation to the pulsed enzyme. The e.p.r. spectrum of the resting enzyme was very similar to that of mitochondrial enzyme, but the transient g = 5, 1.78 and 1.69 set of e.p.r. signals, associated with the pulsed bovine heart oxidase, were not observed in the case of pulsed bacterium-PS3 enzyme.
Characterization of proteoglycans from the calcified matrix of bovine bone
Authors: Franzén A , Heinegård D
Published: August 2015

The proteoglycans characterized were those isolated from the calcified matrix of mature bovine bone [Franzén & Heinegård (1984) Biochem. J. 224, 47-58]. The average molecular mass of the bone proteoglycan is 74 600 Da, determined by sedimentation-equilibrium centrifugation in 4M-guanidinium chloride. Its sedimentation coefficient (s0(20),w) is 3.04 S. The apparent Mr of its core protein is 46 000, estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chondroitinase ABC-digested proteoglycan. A more likely molecular mass of the core protein is 30 000 Da, as calculated from the molecular mass and the protein content (40%) of the proteoglycan. The bone proteoglycan contains one or probably two chondroitin sulphate chains each with a molecular mass (weight-average) of 33 700 Da and several oligosaccharides both of the N-glycosidically and the O-glycosidically linked type. Antibodies against the homogeneous bone proteoglycans were raised in rabbits. An e.l.i.s.a. (enzyme-linked immunosorbent assay) method was developed that allowed specific quantification of bone proteoglycans at nanogram levels. The specificity of the antibodies was tested by using the e.l.i.s.a. method. The bone proteoglycan showed partial cross-reactivity with the small proteoglycan of cartilage. The antibodies were used to localize immunoreactivity of bone proteoglycans by indirect immunofluorescence in frozen sections of foetal bovine epiphysial growth plate. The fluorescence was entirely found in the primary spongiosa, and no fluorescence was found among the hypertrophied chondrocytes or in the region of provisional calcification.
Calcium-ion and calmodulin-dependent κ-casein kinase in rat mammary acini
Authors: Brooks C L , Landt M
Published: August 2015

A Ca2+- and calmodulin-dependent casein kinase specific for dephosphorylated bovine kappa-casein was identified in a microsomal fraction of mammary acini prepared from rats in late lactation. This phosphorylation has an absolute requirement for Mg2+ for either the basal or the Ca2+- and calmodulin-dependent activity. One-half of the maximal stimulation is achieved at a calmodulin concentration of 204nM in the presence of Ca2+. The Ca2+- and calmodulin-dependent kinase activity (but not the basal activity) is inhibited by trifluoperazine. The casein kinase is associated with a microsomal fraction enriched in markers for plasma membrane and Golgi (5′-nucleotidase and galactosyltransferase respectively). The activity of this casein kinase remains relatively constant throughout lactation, but declines dramatically in 24h when rats are removed from their pups. This activity may represent the physiological activity responsible in part or whole for kappa-casein phosphorylation occurring before micelle formation and milk secretion.
Formation of n.m.r.-invisible ADP during renal ischaemia in rats
Authors: Stubbs M , Freeman D , Ross B D
Published: August 2015

Measurement of the adenine nucleotide and inorganic phosphate content of normoxic and ischaemic kidney in vivo has been made, comparing enzymic assay (after freeze-clamping and acid extraction) with quantification by 31P-n.m.r. Both methods give similar results for ATP, and n.m.r. quantification of Pi gives a value 25-50% of that obtained by enzymic assay. ADP, which is largely invisible to n.m.r. in the normoxic kidney, remains invisible during ischaemia despite a 2-3 fold rise in enzymically assayed ADP. N.m.r. and enzymic assay of the acid extracts give similar values for all metabolites measured. The question of ADP binding in the kidney is discussed, as are the implications for the metabolic regulation of ADP-dependent reactions.
Gluconeogenesis from threonine in normal and diabetic rats
Authors: Hetenyi G , Anderson P J , Kinson G A
Published: August 2015

L-[U-14C]Threonine was infused at a steady rate to non-anaesthetized rats starved for 1 or 3 days and to diabetic rats starved for 1 day. The rates of turnover of threonine, calculated from the equilibrium specific radioactivity (SA) of plasma threonine, were 5.79 +/- 1.00, 11.67 +/- 1.43 and 13.35 +/- 1.85 mumol/min per kg body wt. in 1-day-starved, 3-day-starved and diabetic rats respectively. The calculated turnover rate of threonine agreed well with the rate expected from the rate of protein turnover reported in the literature. The equilibrium SA of plasma alanine was 5.1-9.8% of that of threonine in the three groups of rats. The equilibrium SA of glucose was 1.42 and 2.90% of that of threonine in 1-day- and 3-day-starved rats respectively. From the non-equilibrium SA of glucose, it is estimated that a higher percentage of 14C atoms is transferred from threonine to glucose in diabetic than in non-diabetic rats. In spite of increases in gluconeogenesis from threonine in long-starved or diabetic rats, we conclude that threonine remains a minor contributor to plasma glucose. Since it is an essential amino acid, its turnover and contribution to the formation of plasma glucose is an index of catabolism and gluconeogenesis from tissue protein.
Immunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation
Authors: Metsikkö M K , Rajaniemi H J
Published: August 2015

Specific anti-(lutropin receptor) antibodies were produced by immunizing rabbits with lutropin receptor purified from pseudopregnant rat ovary. The anti-receptor serum at 1:100 dilution together with anti-(rabbit gamma-globulin) serum immunoprecipitated 70% of 3H-labelled, purified lutropin receptor and 42% of 125I-chorio-gonadotropin-receptor complex. The antiserum inhibited hormone binding to rat ovarian particles. Pseudopregnant rat ovarian particles were labelled with periodate/NaB3H4 and solubilized with Triton X-100. The Triton X-100 extract was subjected to immunoprecipitation using the anti-receptor serum. When the immunoprecipitate was dissolved and analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate under reducing conditions followed by fluorography, a receptor polypeptide with an apparent Mr 95000 was detected. A receptor down-regulating dose of choriogonadotropin was injected into pseudopregnant rats and their ovaries were removed and homogenized 4 days later, and analysed for immunoprecipitable receptors as above. No receptor molecules were found. Accordingly, the lutropin receptor molecules actually disappear rather than merely become masked from hormone during homologous down-regulation.