Showing papers in "Biochemical Journal in 1978"

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent

Abstract: A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain

Abstract: A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with ‘free’ mitochondria as judged by marker-enzyme assays and electron microscopy.

The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria.

Abstract: The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca2+ was investigated. At 30°C and pH7.0, mitochondria can maintain a steady-state pCa2+0 (the negative logarithm of the free extramitochondrial Ca2+ concentration) of 6.1 (0.8μm). This represents a true steady state, as slight displacements in pCa2+0 away from 6.1 result in net Ca2+ uptake or efflux in order to restore pCa2+0 to its original value. In the absence of added permeant weak acid, the steady-state pCa2+0 is virtually independent of the Ca2+ accumulated in the matrix until 60nmol of Ca2+/mg of protein has been taken up. The steady-state pCa2+0 is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa2+0 is variable and appears to be governed by thermodynamic equilibration of Ca2+ across a Ca2+ uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa2+0 in the region of 6 (1μm-free Ca2+) while accumulating Ca2+. Permeant acids delay the build-up of the transmembrane pH gradient as Ca2+ is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa2+0 of 6. The steady-state pCa2+0 is affected by temperature, incubation pH and Mg2+. The activity of the Ca2+ uniport, rather than that of the respiratory chain, is rate-limiting when pCa2+0 is greater than 5.3 (free Ca2+ less than 5μm). When the Ca2+ electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa2+0 (fall in free Ca2+). At pCa2+0 6.1, the activity of the Ca2+ uniport is kinetically limited to 5nmol of Ca2+/min per mg of protein, even when the Ca2+ electrochemical gradient is large. A steady-state cycling of Ca2+ through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa2+0 by liver mitochondria in vivo.

Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol.

Abstract: 1. The influence of halogen substituents on the 1,2-dioxygenation of catechols was investigated. The results obtained with the two isoenzymes pyrocatechase I and pyrocatechase II from the haloarene-utilizing Pseudomonas sp. B 13 and the pyrocatechase from benzoate-induced cells of Alcaligenes eutrophus B.9 were compared. 2. Substituents on catechol were found to interfere with O2 binding by the two isoenzymes from Pseudomonas sp. B 13, whereas the Km value for catechol kept constant at different O2 concentrations. 3. Electron-attracting substituents decreased the Km values for catechols. 4. Results from binding studies with substituted catechols demonstrated narrow stereospecificities of pyrocatechase I from pseudomonas sp. B 13 and the pyrocatechase from alcaligenes eutrophus B.9. In contrast, a low steric hindrance by substituents in the binding of catechols with pyrocatechase II was observed. 5. Low pK′1 values of substituted catechols resulted in low Michaelis constants. 6. Electron-attracting substituents such as halogen decreased the reaction rates of catechol 1,2-dioxygenation. The correlation of the Vmax. values observed with pyrocatechase II from Pseudomonas sp. B 13 with the substituent constant sigma+ (Okamoto–Brown equation) was distinctly greater than with Hammett9s sigma values. The corresponding logVmax. against sigma+ correlation for pyrocatechase I was considerably disturbed by steric influences of the substituents.

Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues.

Abstract: The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.

Reconstitution of chlorophyllide formation by isolated etioplast membranes.

Abstract: 1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.

Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad.

Abstract: 1. Two catechol 1,2-dioxygenases, pyrocatechase I and pyrocatechase II, were found in 3-chlorobenzoate-grown cells of Pseudomonas sp. B 13. The latter enzyme showed high relative activities with 3- and 4-chlorocatechol compared with catechol. 2. In benzoate-grown cells, only pyrocatechase I was induced. It was purified 29-fold with a final specific activity of 20 mumol of catechol oxygenated/min per mg of protein and an overall yield of 22%. Because of the instability of pyrocatechase II on chromatography and dialysis, no increase of specific activity was obtained during the purification experiments. 3. Molecular weights of pyrocatechase I and pyrocatechase II were 82000 and 67000 respectively. 4. For both pyrocatechases the pH optimum was found to be at 8.0.5. Inhibitions of the two pyrocatechases by Cu2+ and Hg2+ ions and p-chloromercuribenzoate were different. The effect on pyrocatechase I after incubation for 20 h with the heavy metals was decreased by addition of 1 mM-2-mercaptoethanol to the reaction mixture. The inhibition of pyrocatechase II was even enhanced under these conditions. 6. Extradiol cleavage of 3-methylcatechol in addition to intradiol fission at a ratio of 1:14 was observed only with pyrocatechase I.

Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart

Abstract: A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).

Activities and some properties of 5'-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine.

Abstract: 1 The maximal activities of 5′-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues 2 Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, UK,from whom copies can be obtained on the terms indicated in Biochem J (1978), 169, 5 This evidence includes the effects of pH and temperature on the activities of the enzymes 3 In many tissues, the activities of 5′-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo 4 In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase It is suggested that 5′-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration 5 The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Abstract: Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.

The molecular-weight-dependence of the anti-coagulant activity of heparin.

Abstract: It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.

Studies on the orientation of brush-border membrane vesicles

Abstract: Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.

Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture.

Abstract: 1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.

Mechanisms of the ability of insulin to activate the glucose-transport system in rat adipocytes.

Abstract: Isolated rat adipocytes were used to assess the mechanisms of the ability of insulin to accelerate glucose transport. Glucose transport was determined by measuring the initial rates of 2-deoxyglucose uptake, and at 24 degrees C insulin increased the Vmax. of transport from 7.3 +/- 1 to 23.1 +/- 2 nmol/min per 10(6) cells, but the Km value remained unchanged (2.5, cf. 2.4 mM). When the Vmax. of basal and insulin-stimulated transport was measured as a function of temperature (15-37 degrees C), parallel Arrhenius plots were obtained yielding equal activation energies of approx. 59kJ/mol. Since both processes have equal activation energies the data indicate that insulin increases Vmax. by increasing the number of available carriers rather than enhancing intrinsic activity of already functioning carriers. Since the ability of insulin to activate glucose transport did not decrease with temperature (whereas plasma-membrane fluidity declines), it is suggested that lateral diffusion of insulin receptors within the plasma-membrane bilayer is not a rat-determining step in insulin action.

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Abstract: Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.

Articular-cartilage proteoglycans in aging and osteoarthritis.

Abstract: The composition of macroscopically normal hip articular cartilage obtained from dogs of various ages was studied. Pieces of cartilage with signs of degeneration were studied separately. In normal aging, the extraction yield of proteoglycans decreased; the keratan sulphate content of extracted proteoglycans increased and the chondroitin sulphate content decreased. The extracted proteoglycans were smaller in the older cartilage, mainly owing to a decrease in the chondroitin sulphate-rich region of the proteoglycan monomers. The hyaluronic acid-binding region and the keratan sulphaterich region were increased and the molar concentration of proteoglycan probably increase with increasing age. The degenerated cartilage had higher water content and the proteoglycans, as well as other tissue components, gave higher yields. The proteoglycan monomers from the degenerated cartilage were smaller than those from normal cartilage of the same age, and hence had a smaller chondroitin sulphate-rich region and some of the molecules also appeared to lack the hyaluronic acid-binding region. Increased proteolytic activity may be involved in the process of cartilage degeneration.

Physical and chemical properties of human plasma alpha2-macroglobulin.

Abstract: Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

Abstract: The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RCrel, whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNAIle. Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.

Increased calcium-ion influx is a component of capacitation of spermatozoa.

Abstract: Capacitation (modifications required for gamete fusion) is produced by incubating guinea-pig spermatozoa in vitro in a chemically defined medium. It is shown that during such incubation a net uptake of Ca2+ by the sperm occurs in two distinguishable phases. An initial loose association of Ca2+, possibly to surface sites, is unaffected by agents (Mg2+, inhibitors of mitochondiral function) that prevent or delay the exocytotic spermatozoal acrosome reaction. The time course of a secondary Ca2+ uptake parallels or slightly precedes the time course of the acrosome reaction. This parallelism is maintained during a variety of treatments that either expedite (local anaesthetics, ionophore A23187, Triton X-100) or delay (Mg2+, low external Ca2+) the acrosome reaction. We conclude that the secondary Ca2+ influx described herein apparently serves to link alterations of the spermatozoal membrane to subsequent contractile and secretory components of the capacitation sequence.

Resolution of the methane mono-oxygenase of Methylococcus capsulatus (Bath) into three components. Purification and properties of component C, a flavoprotein

Abstract: 1. Ion-exchange chromatography resolves the methane mono-oxygenase from soluble extracts of Methylococcus capsulatus (Bath) into three fractions. 2. Fractions A and B are comparatively stable at 0 degrees C, whereas fraction C is very unstable unless kept in the presence of sodium thioglycollate (1-10 mM) or dithiothreitol (5-10mM). 3. The active component from fraction C was purified some 80-fold. 4. Purified component C has mol. wt. 42000. Its solutions are yellow with absorption maxima at 270 and 465 nm and a shoulder at 395 nm. The 465 nm peak is abolished by reduction with NADH or sodium dithionite, or by photoreduction in the presence of EDTA. A new spectral species, probably a neutral flavin semiquinone, is observed on partial reduction of component C. 5. No copper was detected in samples of purified component C, but the protein contains 1.3-1.5 atoms of iron/molecule. 6. On boiling, component C releases a yellow-green fluorescent material that has been identified as FAD from its absorption and fluorescence spectra and by t.l.c. 7. Component C contains 1 mol of FAD/mol of protein.

Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin).

Abstract: Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.

Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, ribonuclease B and agalacto-orosomucoid.

Abstract: Glycoproteins having mannose and/or N-acetylglucosamine in the terminal non-reducing position [Stockert, Morell & Scheinberg (1976) Biochem. Biophys. Res. Commun. 68, 988--993], and various lysosomal enzymes [Stahl, Schlesinger, Rodman & Doebber (1976) Nature (London) 264, 86--8] are rapidly cleared from plasma by the liver after intravenous administration. A liver cell-separation technique was used to determine the cellular localization of 125I-labelled beta-glucuronidase, ribonuclease B, agalacto-orosomucoid and asialo-orosomucoid. On a specific readioactivity basis, all ligands except 125I-labelled asialo-orosomucoid were enriched in the non-parenchymal cell fraction. Isolated cells, fixed and stained for beta-glucuronidase or N-acetyl-beta-D-glucosaminidase activity after intravenous injection of the enzymes, showed enrichment in the non-parenchymal cell fraction (probably Kupffer cells). After uptake by the non-parenchymal cells, liver lysosomal beta-glucuronidase and N-acetyl-beta-D-glucosaminidase showed degradation half-times of 2.2 and 0.4 days respectively.

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

Abstract: 1 Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing 2 All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack 3 Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength 4 All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem J 128, 1183-1192] of T koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component 5 The mode of action of the enzymes is discussed in relation to these synergistic effects It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

Abstract: 1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.

Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone

Abstract: The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.

Properties of potato lectin and the nature of its glycoprotein linkages

Abstract: 1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer–dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000–34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the β-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although α-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of β-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single α-galactopyranoside residues that can be removed by the action of α-galactosidase from coffee beans but not by a β-galactosidase. This is the first report of an α-galactoside linkage to serine. The effect of α-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of β-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the β-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain β-l-arabinofuranosides linked to hydroxyproline and α-d-galactopyranosides linked to serine residues of the polypeptide chain.

Age-related changes in the composition and structure of human articular-cartilage proteoglycans.

Abstract: 1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety.

Abstract: Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

The interaction of C-protein with heavy meromyosin and subfragment-2.

Abstract: C-protein has previously been shown to bind to the light-meromyosin region of the myosin tail. Examination of mixtures of C-protein with heavy meromyosin or subfragment-2 or subfragment-1 in the analytical ultracentrifuge shows that there is also a binding site for C-protein in the subfragment-2 region of the tail.