Showing papers in "Biochemical Journal in 1983"

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

Abstract: The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

Abstract: The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.

Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat.

Abstract: The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.

Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes.

Abstract: Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.

Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat.

Abstract: The requirement for carnitine and the malonyl-CoA sensitivity of carnitine palmitoyl-transferase I (EC 2.3.1.21) were measured in isolated mitochondria from eight tissues of animal or human origin using fixed concentrations of palmitoyl-CoA (50 microM) and albumin (147 microM). The Km for carnitine spanned a 20-fold range, rising from about 35 microM in adult rat and human foetal liver to 700 microM in dog heart. Intermediate values of increasing magnitude were found for rat heart, guinea pig liver and skeletal muscle of rat, dog and man. Conversely, the concentration of malonyl-CoA required for 50% suppression of enzyme activity fell from the region of 2-3 microM in human and rat liver to only 20 nM in tissues displaying the highest Km for carnitine. Thus, the requirement for carnitine and sensitivity to malonyl-CoA appeared to be inversely related. The Km of carnitine palmitoyltransferase I for palmitoyl-CoA was similar in tissues showing large differences in requirement for carnitine. Other experiments established that, in addition to liver, heart and skeletal muscle of fed rats contain significant quantities of malonyl-CoA and that in all three tissues the level falls with starvation. Although its intracellular location in heart and skeletal muscle is not known, the possibility is raised that malonyl-CoA (or a related compound) could, under certain circumstances, interact with carnitine palmitoyltransferase I in non-hepatic tissues and thereby exert control over long chain fatty acid oxidation.

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Abstract: Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.

Glutamine metabolism in lymphocytes of the rat.

Abstract: The metabolism of glutamine in resting and concanavalin-A-stimulated lymphocytes was investigated. In incubated lymphocytes isolated from rat mesenteric lymph nodes, the rates of oxygen and glutamine utilization and that of aspartate production were approximately linear with respect to time for 60 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 90 min. The major end products of glutamine metabolism were glutamate, aspartate and ammonia: the carbon from glutamine may contribute about 30% to respiration. When both glucose and glutamine were presented to the cells, the rates of utilization of both substances increased. Evidence was obtained that the stimulation of glycolysis by glutamine could be due, in part, to an activation of 6-phosphofructokinase. Starvation of the donor animal increased the rate of glutamine utilization. The phosphoenolpyruvate carboxykinase inhibitor mercaptopicolinate decreased the rate of glutamine utilization by 28%; the rates of accumulation of glutamate and ammonia were decreased, whereas those of lactate, aspartate and malate were increased. The mitogen concanavalin A increased the rate of glutamine utilization (by about 51%). The rate of [3H]thymidine incorporation into DNA caused by concanavalin A in cultured lymphocytes was very low in the absence of glutamine; it was increased about 4-fold at 1 microM-glutamine and was maximal at 0.3 mM-glutamine; neither other amino acids nor ammonia could replace glutamine.

Studies on the mechanism of the antifungal action of benzoate.

Abstract: A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.

A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

Abstract: We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum.

Abstract: The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

Electron-microscopical approach to a structural model of intima collagen.

Abstract: Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S

Abstract: C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.

Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures

Abstract: A fluorographic procedure was optimized which utilized acetic acid as the solvent for 2,5-diphenyloxazole (PPO). This procedure was then compared with existing fluorographic procedures which utilize PPO in dimethyl sulphoxide or sodium salicylate in water, and a commercially available fluorographic solution. En3Hance (New England Nuclear Corp.). A comparison of the four methods revealed that all of the procedures resulted in essentially the same efficiency of radioactivity detection. The acetic acid/PPO procedure was found to have several technical advantages. There is no need to pre-fix proteins in gels, and either agarose or acrylamide gels can be used. The acetic acid/PPO procedure was found to be a simple, sensitive and efficient alternative fluorographic method.

The effect of insulin infusion and food intake on muscle protein synthesis in postabsorptive rats.

Abstract: 1. Insulin was infused into young male rats in the postabsorptive state. Rates of protein synthesis in skeletal muscle were determined during the final 10 min of infusion from the incorporation of label into protein after intravenous injection of a massive dose of [3H]phenylalanine. Rates of synthesis were not altered during the first 10 min of insulin infusion, but were increased significantly between 10 and 60 min. 2. Rats were infused with different amounts of insulin for 30 min. When concentrations were increased from 10 to 40 microunits/ml of plasma there was no change in muscle protein synthesis, but concentrations higher than 70 microunits/ml caused a significant stimulation. Concentrations below 10 microunits/ml, obtained by infusion of anti-insulin serum, did not depress synthesis below that found in the postabsorptive rat. 3. Infusion of glucose for 30 or 60 min led to an increase in plasma insulin to 40 microunits/ml, but this also failed to stimulate muscle protein synthesis. 4. Rates of synthesis in postabsorptive rats, even when stimulated maximally by insulin, were not so high as those in fed rats or in postabsorptive rats refed for 60 min. However, in fed and refed rats insulin concentrations were below that required to stimulate synthesis in postabsorptive animals. Despite this, infusion of large amounts of insulin into fed rats did not increase synthesis further. 5. The sensitivity of plasma glucose to insulin infusion was different from that of protein synthesis. A decrease in glucose concentration preceded the increase in synthesis and occurred at lower insulin concentrations. 6. It is concluded that changes in circulating insulin may have been partly responsible for the increase in muscle protein synthesis brought about by feeding, but that other factors must also play a part.

High resolution 1H n.m.r. studies of vertebrate blood and plasma.

Abstract: Spin echo Fourier transform proton n.m.r. spectra of whole blood contain resonances from both erythrocytes and plasma. A large number of well-resolved signals from mobile protons of low-molecular-weight metabolites in plasma and serum have been identified. Spectra from the plasmas of eight animal species and commercial, quality control sera are compared. CaEDTA2- and MgEDTA2- resonances can be used for the simultaneous determination of EDTA-chelatable calcium and magnesium concentrations in intact plasma and other biological fluids. Cholesterol is too immobile to contribute to the spectra of intact plasma, but is readily estimated by n.m.r. in both its free and esterified forms after extraction into methanol.

Regulation of immunoreactive-insulin release from a rat cell line (RINm5F)

Abstract: 1 An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied 2 The cellular content of immunoreactive insulin was 019 pg/cell, which represents approx 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively 3 RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 28 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer 4 Glucose utilization was increased by raising glucose from 28 to 167 mM There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 28 to 334 mM A small stimulation of release was, however, found when glucose was raised from 0 to 28 mM 5 Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner 6 At 20 mM, leucine or arginine stimulated release at 28 mM-glucose 7 Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 28 mM-glucose with no additional stimulation at 167 mM-glucose 8 Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM Another depolarizing agent, ouabain, also stimulated release 9 Adrenaline (epinephrine) inhibited both basal (28 mM-glucose) release and that stimulated by 30 mM-K+ 10 Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 03 or to 01 mM-Ca2+ lowered the release 11 These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release

Structure of proteoglycans from different layers of human articular cartilage.

Abstract: Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree)

Abstract: Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa.

Isolation and characterization of human cervical-mucus glycoproteins

Abstract: Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

Measurement of specific radioactivities in labelled hormones by self-displacement analysis.

Abstract: A graphical method is described that allows the determination of specific radioactivities of radioactively labelled hormones. This method combines the self-displacement technique, plotting bound/free ratios versus mass of unlabelled hormone or total radioactivity of labelled preparation added to the receptor preparation, and the maximal binding capacity of the labelled hormone. The procedure presented herein provides a more realistic specific radioactivity for use in all binding experiments. Application of the method is demonstrated for 125I-labelled ovine prolactin, and data are presented for 125I-labelled human choriogonadotropin and [3H]testosterone.

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

Abstract: The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.

Endocytosis: a review of mechanisms and plasma membrane dynamics.

Abstract: Endocytosis is the process of internalization of extracellular material within an invagination of the plasmalemma. Ultimately, this invagination is thought to give rise to an independent cytoplasmic vesicle. This vesicle is given a more specific name (e.g. phagosome, pinosome, coated vesicle, primary food vacuole) depending on the cell type being studied and the mode of endocytosis involved. Historically, the multitude of morphological varieties of endocytosis has led to a plethora of confusing terminology (e.g. cytosis, endocytosis, intracytosis). However, it was de Duve in 1963 who used the term 'endocytosis' to encompass all variant forms of this basic cellular transport mechanism. Classically, endocytosis has been divided into two types: phagocytosis, or eating, and pinocytosis, or drinking. The term phagocytosis is used to describe the internalization of particulate matter visible by light microscopy and, hence, larger than a few tenths of a pm in diameter. The term pinocytosis is used to describe the uptake of all smaller substrates, ranging from insoluble particles to low molecular weight solutes and to the fluid itself. Pinocytosis can be subdivided further, based on whether the substance enters the cell in the fluid content (fluid-phase pinocytosis) or bound to the vesicle membrane (adsorptive pinocytosis). Moreover, adsorptive pinocytosis can be subdivided further into endocytosis of markers which bind in a non-specific manner to the cell surface (e.g. cationic ferritin binding to anionic sites on the plasmalemma), and endocytosis of ligands mediated via binding to specific receptors on the cell membrane. The latter mechanism has been called receptor-mediated endocytosis (pinocytosis). Molecules known to be taken up by this receptormediated mechanism include lysosomal enzymes

Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components.

Abstract: A metalloproteinase, 'proteoglycanase', that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the 'weak region' of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.

Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver

Abstract: The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.

Ca2+-dependent high-affinity complex formation between calmodulin and melittin.

Abstract: The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.

Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate

Abstract: The concentrations of Zn2+, Ca2+, Mg2+, Pi and adenine nucleotides were determined in insulin-secretory granules prepared from a transplantable rat insulinoma. Differential and density-gradient centrifugation analyses revealed that Zn2+ in this tissue was principally localized in the secretory granule, a second major fraction being found in association with cytosolic proteins. Pi was principally recovered in the latter fraction, whereas Ca2+ and Mg2+ were more widely distributed. Intragranular ion-distribution experiments suggested that Zn2+ was complexed mainly to insulin and its precursor forms and remained in the granule in an insoluble state. The Zn2+/insulin ratio (0.54) was greater than that expected for insulin molecules having two centrally co-ordinated Zn2+ atoms/hexamer, but less than the maximal Zn2+-binding capacity of the molecule. Most of the granular Ca2+, Mg2+ and Pi was released in a soluble form when granules were disrupted by sonication. Simulation in vitro of the ionic composition of the granule suggested that up to 90% of its Ca2+ was complexed to Pi and adenine nucleotides. Granular macromolecules also bound Ca2+, as shown by equilibrium-dialysis studies of granule lysates. However, such binding was displaced by Mg2+. Examination of the efflux of Ca2+ from granules incubated in iso-osmotic suspensions at 37 degrees C suggested that the passive permeability of the granule membrane to Ca2+ was very low. Nevertheless, more than 50% of the granular Ca2+ was rapidly released in an ionized form on hypo-osmotic or detergent-induced disruption of the granule membrane. This may represent a potentially mobilizable pool of Ca2+ in vivo.

Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action.

Abstract: Thyrotropin-releasing hormone (TRH; thyroliberin) stimulated rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phosphodiesterase (phospholipase C) in GH3 cells, a prolactin-secreting rat pituitary tumour cell line. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 60% of control and stimulated a marked transient increase in inositol 1,4,5-trisphosphate, the unique product of phosphodiesteratic hydrolysis of PtdIns(4,5)P2, to a peak of 410% of control at 15 s. TRH also caused decreases in phosphatidylinositol 4-monophosphate (PtdIns4P) and phosphatidylinositol (PtdIns) to 65% and 93% of control at 15 s respectively. Inositol 1,4-bisphosphate was increased to a peak of 450% at 30 s; inositol 1-monophosphate and inositol were not elevated until 30 s and 1 min respectively after TRH addition. To study whether PtdIns(4,5)P2 hydrolysis may be caused by an elevation in cytosolic Ca2+ concentration, the changes induced by TRH in the levels of inositol sugars were compared with the effects of membrane depolarization by high extracellular [K+]. The elevation in cytosolic [Ca2+] induced by K+ depolarization did not change the level of inositol 1,4,5-trisphosphate. These data suggest that phosphodiesteratic hydrolysis of PtdIns(4,5)P2 may be the initial event in TRH stimulation of inositol lipid metabolism in GH3 cells and that PtdIns(4,5)P2 hydrolysis is not stimulated by an elevation in cytosolic Ca2+ concentration. The decreases in PtdIns4P and PtdIns may be due to enhanced conversion of PtdIns into PtdIns4P into PtdIns(4,5)P2 or to their direct hydrolysis by phosphomonoesterases and/or phosphodiesterases. These results are consistent with the hypothesis that TRH-stimulated PtdIns(4,5)P2 breakdown causes Ca2+ mobilization leading to prolactin secretion.

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth.

Abstract: Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5′-nucleotidase are suggested as modulators of hyaluronate synthesis.

Rhodamine-based compounds as fluorogenic substrates for serine proteinases.

Abstract: A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.