bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin
Authors: Van Leuven Fred , Cassiman Jean-Jacques , Van Den Berghe Herman
Published: August 2015

The unique steric inhibition of endopeptidases by human α2M (α2-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by α2M was closely correlated with the loss of the methylamine-reactive sites in α2M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/α2M ratios, no unaccounted loss of trypsin-binding capacity occurred. As α2M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form α2M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [14C]methylamine with α2M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of α2M with time of methylamine treatment. It was found that conformational change of α2M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of 125I-labelled trypsin to α2M did follow the same kinetics as the conformational change. This discrepancy between total binding (125I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound 125I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of α2M and to the mechanism of formation of the complex.
Photolytic studies on the carbon monoxide complex of sulphaemoglobin
Authors: Brittain T , Greenwood C
Published: August 2015

The CO complex of sulphaemoglobin was found to be photolytically dissociable at pH 6.0 and pH 8.5. The recombination kinetics of the CO complex after flash photolysis show two phases, which differ in rate by approximately 20-fold. At both pH 6.0 and pH 8.5 the two phases show an identical linear dependence on CO concentration, with associated second-order rate constants of 6.0 × 10(3) M-1 . s-1 and 1.2 × 10(5) M-1 . s-1 respectively. The percentage contribution of each of the two phases to the total absorbance change is independent of the CO concentration employed, but is a function of the protein concentration used. The percentage of the faster phase increases with protein dilution. These results taken together with computer simulation suggest the existence of appreciable amounts of monomeric species in solution at both pH 6.0 and pH 8.5 at low concentration of liganded protein. Estimates of the dissociation constants for the dimer in equilibrium monomer equilibrium yield values of 8 microM and 3.5 microM at pH 6.0 and pH 8.5 respectively. Stopped-flow delayed-flash-photolysis studies show that the liganded dimer decays to the liganded monomer with a half-life of approx. 0.5 s. Constant-illumination experiments allow an estimation of the half-life for the combination of deoxy monomers to deoxy dimers of 22 s. A model explaining the range of CO recombination kinetics seen for sulphaemoglobin does not require the existence of a photolytically produced high-activity form, even at high pH, in contrast with the case for normal haemoglobin.
Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings
Authors: Noguchi T , Fujiwara S
Published: August 2015

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.
Ion-exchange chromatography in the presence of the non-ionic dissociating agent chloral hydrate. Application to a membrane protein, bovine heart cytochrome c oxidase
Authors: Griffin D C , Landon M
Published: August 2015

In previous work we have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) is a potent non-ionic protein dissociating agent. We have employed it in systems of polyacrylamide-gel electrophoresis and have demonstrated the presence of 15 components in a preparation of bovine heart cytochrome c oxidase [Griffin & Landon (1981) Biochem. J. 197, 333-344]. Here we describe the use of solutions containing aq. 100% (w/v) chloral hydrate in the ion-exchange column chromatographic separation on CM-cellulose of the alpha- and beta-chains of human haemoglobin, which we have employed as a model protein of known structure. We also describe the use of similar procedures in order to fractionate the polypeptide components of bovine heart cytochrome c oxidase. An effective separation has been obtained and we suggest that chloral hydrate-containing solutions could have general application in the ion-exchange-chromatographic analysis of membrane proteins, a procedure that has had restricted use owing to the inadequacy of non-ionic dissociating agents available previously.
Evidence from electron-paramagnetic-resonance spectroscopy for a complex of sulphite ions with the molybdenum centre of sulphite oxidase
Authors: Bray R C , Lamy M T , Gutteridge S , Wilkinson T
Published: August 2015

Reduction of sulphite oxidase by sulphite at low pH values in Mes (4-morpholine-ethanesulphonic acid) buffer gives rise to a new molybdenum(V) electron-paramagnetic-resonance spectrum different from that obtained by photoreduction of the enzyme in the same medium. The spectrum is attributed to a sulphite complex of the enzyme, showing g-values of about 2.000, 1.972 and 1.963. The complex is analogous to that with the inhibitor phosphate in that it gives rise to no observable hyperfine coupling of Mo(V) to exchangeable protons.
Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.
Authors: Uotila L , Suttie J W
Published: August 2015

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.
Hereditary spherocytosis of man. Altered binding of cytoskeletal components to the erythrocyte membrane
Authors: Hill John S. , Sawyer William H. , Howlett Geoffrey J. , Wiley James S.
Published: August 2015

Human erythrocytes possess a lattice work of extrinsic proteins on the inner face of the membrane (‘cytoskeleton’) that maintains the shape and deformability of the cell. The major proteins of the cytoskeleton are spectrin and actin, which are attached to the membrane by protein bands 2.1 (‘ankyrin’) and 4.1. The interactions of spectrin/actin with erythrocyte membranes from normal subjects and from patients with hereditary spherocytosis (HS) have been studied by using an air-driven ultracentrifuge, which can rapidly separate membranes from soluble proteins (150000g for 30s). The total amount of spectrin/actin in HS and normal ghosts is similar. However, the rate of dissociation of spectrin and actin from HS erythrocyte membranes at low ionic strength is significantly lower than that observed for normal membranes. Spectrin and actin isolated from either HS or normal membranes re-associated in a similar manner to spectrin/actin-depleted vesicles prepared from normal cells. Scatchard analysis showed an average binding capacity of 278μg/mg of membrane protein. However, spectrin/actin-depleted vesicles prepared from HS cells bound significantly less spectrin/actin prepared from either the normal or abnormal cells (average binding capacity 158μg/mg of membrane protein). The defect was defined further by studying the cytoskeleton obtained by Triton X-100 extraction of membranes. Under conditions of low ionic strength cytoskeletons prepared from HS membranes dissociated more slowly than those prepared from normal membranes, and only 80% of the protein from HS cytoskeletons could be solubilized after 180min compared with 100% for normal cytoskeletons. The difference between HS and normal membranes, which persists in isolated cytoskeletons, suggests that alterations in either the primary structure or the degree of phosphorylation of protein bands 2.1 or 4.1 may be central to the molecular basis of hereditary spherocytosis.
Characterization of immunoreactive somatostatin released from rat nervous system in vitro.
Authors: Sheppard M C , Hendricks S , Hudson A , Kronheim S R
Published: August 2015

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, ‘STH’, ‘GH’) release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.
The stereochemical course of phosphoryl transfer catalysed by glucokinase.
Authors: Pollard-Knight D , Potter B V L , Cullis P M , Lowe G , Cornish-Bowden A
Published: August 2015

Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an ‘in-line’ mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.
Slow structural changes shown by the 3-nitrotyrosine-237 residue in pig heart [Tyr(3NO2)237] lactate dehydrogenase
Authors: Parker D M , Jeckel D , Holbrook J J
Published: August 2015

1. The pKa of the phenolic hydroxy group of the Tyr(3NO2)-237 residue in pig heart [Tyr(3NO2)237]lactate dehydrogenase is 7.2 in the apoenzyme, 7.4 in the enzyme-NADH complex and 7.8 in the enzyme-NADH-oxamate complex. The alkaline shift from apoenzyme to ternary complex is ascribed to the approach of the Glu-107 residue during the movement of the polypeptide loop residues 98-110. 2. The affinities of the nitrated enzyme for NADH and for oxamate (in the presence of NADH) are slightly less than those of the native enzyme. The turnover number for the nitrated enzyme in the pyruvate-to-lactate direction is about 0.75 of the value for the native enzyme. 3. Temperature-jump relaxation experiments of the enzyme saturated with NADH but fractionally saturated with oxamate are interpreted to show that the pKa of the nitrotyrosine residue responds to a protein rearrangement after oxamate binds to the binary enzyme-NADH complex. 4. Transient-kinetic experiments show the environment of the Tyr(3NO2)-237 residue in the enzyme-NADH-pyruvate complex of the steady state to be similar to that in the enzyme-NADH-oxamate inhibitor complex.
The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous
Authors: Cheetham P S J , Dunnill P , Lilly M D
Published: August 2015

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.
Studies of the conformation of bilirubin and its dimethyl ester in dimethyl sulphoxide solutions by nuclear magnetic resonance
Authors: Kaplan D , Navon G
Published: August 2015

The conformation of bilirubin and its dimethyl ester in dimethyl sulphoxide (DMSO) was investigated by n.m.r. spectroscopy. The chemical shifts of the pyrrole NH and Lactam protons of bilirubin and its dimethyl ester in DMSO indicate a strong interaction with the solvent. Inter-proton distances were calculated from nuclear Overhauser effects (NOE), selective and non-selective relaxation times (T1) and rotational correlation times taken from 13C relaxation times. The interproton distances indicate that the conformation of the skeleton of bilirubin and its dimethyl ester in DMSO is similar to that of bilirubin and mesobilirubin in the crystalline state and in chloroform solutions, except for a possible slight twist of the pyrrolenone rings about the methine bonds, which may be a consequence of solvation of the NH groups by DMSO. Unlike in chloroform solutions, no direct hydrogen-bonding occurs between the carboxylic acid and the lactam groups of bilirubin in DMSO, as shown by the absence of an NOE between these groups. The fast exchange of the pyrrole NH protons with 2H shows that no hydrogen-bonding occurs between these protons and the propionic residues, in line with their solvation by DMSO. From the above results, and from the slowness of the internal motion of the propionic residues of bilirubin and its dimethyl ester, it is concluded that these residues are tied to the skeleton via bound solvent molecules.
Solubilization and partial characterization of rat epididymal Δ 4-steroid 5 α-reductase (cholestenone 5 α-reductase)
Authors: Scheer H , Robaire B
Published: August 2015

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 × 10(-7) × 4.52 × 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.
Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein
Authors: Sedlmeier D , Dieberg G
Published: August 2015

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.
CO-binding c-type cytochromes and a high-potential cytochrome c in Nitrosomonas europaea
Authors: Miller D J , Wood P M
Published: August 2015

The purification of two soluble CO-binding cytochromes c from Nitrosomonas europaea is described. Cytochrome cCO−550 ran on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 32 000, whereas for cytochrome cCO−552 the apparent Mr was 16 000. Redox potentials (Em, 7) were determined as +140 and −50mV respectively. Cytochrome cCO−550 was co-purified with a cytochrome c−553, for which an unusually high redox potential of +450mV was measured. These latter components were not resolved by gel-filtration chromatography or electrophoresis under denaturing conditions.
Action of cathepsin D on fructose-1,6-bisphosphate aldolase
Authors: Offermann M K , Chlebowski J F , Bond J S
Published: August 2015

Cathepsin D inactivated aldolase at pH values between 4.2 and 5.2; the chloride, sulphate or iodide, but not citrate or acetate, salts of sodium or potassium accelerated the rate of inactivation. Cathepsin D cleaved numerous peptide bonds in the C-terminus of aldolase, but the major site of cleavage in this region was Leu354-Phe355. The most prominent peptide products of hydrolysis were Phe-Ile-Ser-Asn-His-Ala-Tyr and Phe-Ile-Ser-Asn-His. Up to 20 amino acids were removed from the C-terminus of aldolase, but no further degradation of native aldolase was observed. By contrast, extensive degradation of the 40 000-Mr subunit was observed after aldolase was denatured. The cathepsin D-inactivated aldolase cross-reacted with antibodies prepared against native aldolase and had the same thermodynamic stability as native aldolase, demonstrated by differential scanning calorimetry and fluorescence quenching of tryptophan residues. Furthermore, the cathepsin-modified and native forms of aldolase were both resistant to extensive proteolysis by other purified cellular proteinases and lysosomal extracts at pH values of 4.8-8.0.
The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur
Authors: Williamson G , Engel P C
Published: August 2015

Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. 5,5′-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. 1 mol/mol of FAD. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. The enzyme remains fully active. Phenylmercuric acetate also de-greens the enzyme. The extent to which thiols restore the green colour declines with time. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. The results are explicable if it is assumed that the thionitrobenzoate derivative of CoA persulphide loses the extra sulphur atom much more readily than does the phenylmercury derivative.
Isolation and characterization of the pig endometrial arylsulphatase A.
Authors: Rahi H , Srivastava P N
Published: August 2015

The pig endometrial arylsulphatase A was purified 3322-fold to a specific activity of 150 mumol/min per mg. The purification involved (NH4)2SO4 fractionation, chromatography on concanavalin A-Sepharose and DEAE-Sepharose, gel filtrations on Sephadex G-200 at pH 7.4 and 5, and a new preparative gel-electrophoresis technique. The homogeneous enzyme is a glycoprotein containing 20% carbohydrate. The purified enzyme has Mr about 120 000 and it contains subunits of Mr 63 000. The pig endometrial arylsulphatase A shows many properties in common with those of arylsulphatases A purified from other sources. The similarities include their low isoelectric points, the anomalous time-activity relationships, multi-pH optima, inhibition by SO3(2-), SO4(2-), phosphate ions, metal ions and nucleoside phosphates, pH- and ionic-strength-dependent polymerization and amino acid composition.
Studies by e.p.r. spectroscopy of carbon monoxide oxidases from Pseudomonas carboxydovorans and Pseudomonas carboxydohydrogena
Authors: Bray R C , George G N , Lange R , Meyer O
Published: August 2015

E.p.r. spectra were obtained at 8-120 K for carbon monoxide oxidases isolated from the carboxydotrophic bacteria Pseudomonas carboxydovorans and Pseudomonas carboxydohydrogena. Spectra from the two enzymes are extremely similar to one another. Under appropriate conditions each enzyme shows signals from Mo(V) atoms in two different chemical environments, as well as showing signals from two distinct iron-sulphur centres, presumed to be [2Fe-2S] clusters, and weak FADH X free-radical signals. Parameters of most of the signals were measured, and they show considerable similarities to those of the corresponding signals from xanthine oxidase and related enzymes. Though the signals from carbon monoxide oxidases appear and disappear under reducing and oxidizing conditions, we have so far failed to demonstrate the kinetic competence of any of them. It seems likely that this was due to the presence in the enzyme preparation examined of high amounts of desulpho carbon monoxide oxidase together with another non-functional form of the enzyme giving a stable ‘Resting’ Mo(V) e.p.r. signal.
Isolation and characterization of a trypsin-like serine proteinase from the membranes of Walker 256 carcino-sarcoma cells
Authors: LaBombardi V J , Shaw E , DiStefano J F , Beck G , Brown F , Zucker S
Published: August 2015

A serine proteinase was isolated from Walker-256-carcino-sarcoma plasma-membrane-enriched preparations by affinity chromatography employing soya-bean trypsin inhibitor as the ligand. This enzyme was termed ‘memsin’ owing to its membrane location and trypsin-like substrate specificity. Analysis of this preparation by steric-exclusion high-pressure liquid chromatography (h.p.l.c.) resulted in a single peak of enzyme activity. Calculations of the rates of inactivation of memsin by peptidyl-chloromethanes and comparison with rate constants obtained with other serine proteinases indicated that memsin closely resembled trypsin and acrosin. Digestion of oxidized ribonuclease by memsin and analysis of the resulting peptides by h.p.l.c. yielded a chromatogram that was very similar to one generated by a tryptic digest of oxidized ribonuclease. This enzyme could possibly play a role in tumour-cell invasion.
Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein
Authors: Kenny A J , Fulcher I S , McGill K A , Kershaw D
Published: August 2015

Pig kidney microvillar proteins were extracted with octyl β-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, ‘right-side-out’, orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.
Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum
Authors: Cercek B , Wilson S R , Houslay M D
Published: August 2015

A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.
Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors
Authors: Glass D B
Published: August 2015

The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.
A study of factors influencing hydration of sodium hyaluronate from compressibility and high-precision densimetric measurements
Authors: Davies A , Gormally J , Wyn-Jones E , Wedlock D J , Phillips G O
Published: August 2015

A study of the factors influencing the hydration of the biopolymer hyaluronic acid was made by compressibility and density measurements. The factors investigated were the hydration changes on glycosidic bond formation, and also the influence of counterion type, solution ionic strength and temperature. The results indicate that, with this biopolymer, the hydration of the glucuronate residue is significantly more than that of the N-acetylglucosamine residue, and further that the biopolymer is less hydrated than the sum of its component monosaccharide residues. Change of the counterion salt form of this polyelectrolyte from univalent to bivalent counterion type (Na+ to Ca2+) leads to a small though significant increase in the total hydration sheath surrounding the polymer. An increase in the background ionic strength of the solvent leads to a quantifiable lowering of the hydration of the polymer at physiological ionic strength compared with its value in salt-free aqueous solution. A decrease in hydration with increase in temperature in the range 20-50 degrees C is the opposite of previous reports, and was observed when the polymer was dissolved both in pure water and in 0.15 M-NaCl.
Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells
Authors: Strewler G J , Danello M A , Manganiello V C , Vaughan M
Published: August 2015

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.
Action of human liver cathepsin B on the oxidized insulin B chain
Authors: McKay M J , Offermann M K , Barrett A J , Bond J S
Published: August 2015

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.
Role of membrane-associated thiol groups in the functional regulation of gastric microsomal (H+ + K+)-transporting ATPase system
Authors: Nandi J , Meng-Ai Z , Ray T K
Published: August 2015

The distribution of free thiol groups associated with the membrane proteins of the purified pig gastric microsomal vesicles was quantified, and the relation of thiol groups to the function of the gastric (H+ + K+)-transporting ATPase system was investigated. Two different thiol-specific agents, carboxypyridine disulphide (CPDS) and N-(1-naphthyl)maleimide (NNM) were used for the study. The structure-function relationship of the membrane thiol groups was studied after modification by the probes under various conditions, relating the inhibition of the (H+ + K+)-transporting ATPase to the ATP-dependent H+ accumulation by the gastric microsomal vesicles. On the basis of the extent of stimulation of the microsomal (H+ + K+)-transporting ATPase in the presence and absence of valinomycin (val) about 85% of the vesicles were found to be intact. CPDS at 1 mM completely inhibits the valinomycin-stimulated ATPase and the associated p-nitrophenyl phosphatase with a concomitant inhibition of vesicular H+ uptake. Both the enzyme and dye-uptake activities were fully protected against CPDS inhibition when the treatment with CPDS was carried out in the presence of ATP. ATP also offered protection (about 65%) against NNM inhibition of the (H+ + K+)-transporting ATPase system and vesicular H+ uptake. Under similar conditions ATP also protected about 10 and 6 nmol of thiol groups/mg of protein respectively from CPDS and NNM reaction. Our data suggest that the thiol groups on the outer surface of the vesicles are primarily involved in gastric (H+ + K+)-transporting ATPase function. Furthermore, at least about 15% of the total microsomal thiol groups appear to be associated with the ATPase system. The data have been discussed in terms of the structure-function relationship of gastric microsomes.
Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate
Authors: Leadlay P F , Fuller J Q
Published: August 2015

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.
An extended X-ray-absorption-fine-structure study of the copper and zinc sites of freeze-dried bovine superoxide dismutase
Authors: Blackburn N J , Hasnain S S , Diakun G P , Knowles P F , Binsted N , Garner C D
Published: August 2015

Copper and zinc K-edge-extended X-ray-absorption fine structures were measured for the metal sites of freeze-dried bovine superoxide dismutase and the model compounds tetrakis(imidazole)cupric nitrate and tetrakis(imidazole)zinc perchlorate. Detailed simulation of the spectra indicates that the copper site of the enzyme is best fit by co-ordination of four imidazole groups with Cu-N(alpha) distances of 0.198 nm (1.98 A). The zinc site is best fit by three imidazole groups at 0.201 nm (2.01 A) and an oxygen (from aspartate) at 0.203 nm (2.03 A).
The brain isoform of a key ATP-regulating enzyme, creatine kinase, is a phosphoprotein
Authors: Mahadevan L C , Whatley S A , Leung T K C , Lim L
Published: August 2015

Two-dimensional electrophoretic analysis of crude microtubule preparations from the rat brain revealed the presence of three polypeptides in positions corresponding to those of the isovariants of purified rat brain creatine kinase (CK-BB). By the use of [gamma-32P]ATP, the two more acidic forms of these polypeptides were shown to be phosphorylated. Their identity as phosphorylated forms of CK-BB was established by using various peptide mapping techniques. Thus CK-BB is a phosphoprotein and its isoelectric variation may be attributed to phosphorylation.
Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor
Authors: Berridge M J , Heslop J P , Irvine R F , Brown K D
Published: August 2015

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in ‘leaky’ cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from ‘leaky’ cells, which were insensitive to PDGF. ‘Leaky’ cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.
Phosphorylation of a stromal enzyme protein in maize (Zea mays) mesophyll chloroplasts
Authors: Foyer C
Published: August 2015

When intact maize (Zea mays) mesophyll chloroplasts were illuminated in the presence of [32P]orthophosphate and subsequently subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a major polypeptide species of Mr 100000 was found to be heavily labelled. This polypeptide was not found in maize mesophyll thylakoid or cytoplasmic fractions, but was localized solely in the chloroplast stroma. No phosphorylation of polypeptides in the 100000-Mr region was observed in the mesophyll chloroplasts from C3 species (where the primary product of CO2 fixation is a 3-carbon compound), suggesting that this polypeptide arises from a protein associated with C4 metabolism (where the first product of CO2 fixation is a 4-carbon compound). The 100kDa polypeptide was major component of the maize mesophyll chloroplast, comprising 10-15% of the total protein, which banded in an identical position to the apoprotein of the enzyme pyruvate, orthophosphate dikinase, which catalyses a reaction of the C4 cycle [Edwards & Walker (1983) C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis, Blackwell Scientific Publications, Oxford and London]. Phosphorylation in the 100kDa species was prohibited by treatment of lysed chloroplasts with antibody to pyruvate, orthophosphate dikinase (EC 2.7.9.1). These data suggest that the phosphorylated polypeptide observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is the monomeric form of this enzyme. The 100kDa polypeptide was partially phosphorylated in darkness, but a significant increase in the degree of phosphorylation was found on illumination. This polypeptide was found to be dephosphorylated only slowly when the chloroplasts were returned to darkness. Maximum phosphorylation was observed in the presence of pyruvate or dihydroxyacetone phosphate, which also caused maximum activation of pyruvate, orthophosphate dikinase. Phosphorylation of the 100kDa polypeptide did not coincide with deactivation of pyruvate, orthophosphate dikinase, but maximum phosphorylation occurred under conditions that promoted maximum activity of the enzyme, at which time one phosphate group was associated with each enzyme molecule. Protein phosphorylation did not appear to arise from the reaction mechanism of the enzyme.
Specificity of inositol trisphosphate-induced calcium release from permeabilized Swiss-mouse 3T3 cells
Authors: Irvine R F , Brown K D , Berridge M J
Published: August 2015

Swiss-mouse 3T3 cells permeabilized with saponin were used to study the specificity of the inositol trisphosphate-induced release of 45Ca2+ from their intracellular stores. Inositol 1,4,5-trisphosphate was the most potent compound studied (dose giving half-maximal effect 0.3 microM). 45Ca2+ was also released by inositol 2,4,5-trisphosphate, glycerophosphoinositol 4,5-bisphosphate and inositol 4,5-bisphosphate, with doses giving half-maximal effect of respectively 1.6 microM, 1.6 microM and 20 microM, but not by inositol 1,4-bisphosphate (50 microM). These data suggest that the trans-vicinal phosphates on the 4- and 5-positions are essential for the Ca2+-mobilizing effect of inositol trisphosphate, and that in addition there is a requirement for a phosphate group on the opposite side of the molecule, with a preference for the 1-position.