bluuebunny/crossref_metadata_2025 huggingface dataset:
A Genetic Approach to Reveal the Action of the Opiate Receptor in Selected Neuroblastoma‐Glioma Cells
Authors: SIMANTOV Rabi , NADLER Hana , LEVY Rivka
Published: July 2010

Three clones of neuroblastoma‐glioma cells that contain low amounts of calmodulin were selected from the NG108‐15 cells after several treatments with high concentrations of chlorpromazine. Purified membranes of the three clones had decreased numbers of both α‐adrenergic and opiate receptors, monitored with [3H]yohimbine and [3H,d‐Ala2]methionine encephalinamide, respectively. No changes were observed in the affinity of these radioactive ligands to the receptors of the selected cells as compared to the parent cells. Addition of bovine brain calmodulin did not affect the binding of [3H,d‐Ala2]methionine encephalinamide to the membranes of the selected cells and they had the same number of acetylcholine receptors, determined with 1‐quinuclidinyl‐[phenyl‐4‐3H]‐benzilate, as the parent NG108‐15 cells.

The basal ATPase activity in the membranes of the selected cells was 35–50% of the parent cells, with a decreasedVvalue and no significant change in the affinity constantKato ATP. Addition of Ca2+to the purified membranes increased theVof the ATPase in the selected as well as the parent cells but theVof the selected cells remained lower than that of the parent cells. Ca2+had no effect on theKato ATP in either cell type. The Ca2+‐dependent ATPase activity of both the parent and the selected cells was also calmodulin‐dependent since it was blockedin vitroby chlorpromazine. The co‐regulation of opiate and adrenergic receptors and their interaction with calmodulin and Ca2+‐ATPase is discussed in view of recent observations indicating biochemical and physiological association between opiates, Ca2+and adrenergic compounds.
Wild‐Type and Mutant Forms of Homoisocitric Dehydrogenase in the Yeast Saccharomycopsis lipolytica
Authors: GAILLARDIN Claude M. , RIBET Anne‐Marie , HESLOT Henri
Published: July 2010

Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525‐fold from the yeast Saccharomycopsis lipolytica with a yield of 25%. The preparation was judged to be homogeneous by electrophoresis under denaturing and non‐denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme.

Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10). Lvs10 mutants displayed NAD‐reducing activity, whereas lys9 mutants retained some carboxylating activity.

Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.
Primary Gene Products of Bovine β‐Crystallin and Reassociation Behavior of Its Aggregates
Authors: BERBERS Guy A. M. , BOERMAN Otto C. , BLOEMENDAL Hans , DE JONG Wilfried W.
Published: July 2010

β‐Crystallin from calf lens cortex was fractionated in three different aggregates of increasing size: βL2, βL1 and βH, of which the subunit composition was revealed by 2‐dimensional gel electrophoresis. While βL2 mainly consists of βBP, (the major polypeptide chain in all three aggregates), βL1 is characterized by the addition of a neutral and two acidic chains, and βH contains moreover two basic chains. Translation of calf lens polyribosomes in a reticulocyte cell‐free system allowed the identification of six β‐crystallin subunits as primary gene products. The distribution of these newly synthesized polypeptides over the three aggregates was established after gel filtration in the presence of carrier lens proteins. The aggregation behavior of the β‐crystallin chains was studied by dissociation/reassociation experiments. The three separate aggregates could be reversibly dissociated. Reassociation of basic, neutral and acidic polypeptides, isolated by ion‐exchange chromatography of β‐crystallin, produced a βH‐like aggregate. The neutral and acidic polypeptides reassociated into a βL1‐like aggregate, while the neutral polypeptides gave dimers like βL2. A βH‐like aggregate could also be obtained by reaggregation of βL2 with the acidic and basic chains of βH. On the basis of these results a preliminary model for the formation of β‐crystallin aggregates is discussed.
Classification of Rat Lens Crystallins and Identification of Proteins Encoded by Rat Lens mRNA
Authors: RAMAEKERS Frans , DODEMONT Huub , VORSTENBOSCH Pieter , BLOEMENDAL Hans
Published: July 2010

Endogenous rat lens crystallins have been separated by gel filtration into four fractions, α, βH, βL and γ‐crystallin. Elution patterns of soluble lens proteins from animals of different ages show a relative decrease of βH and γ‐crystallin during aging. Conversely the relative amounts of α and βL‐crystallin are enhanced. The rat crystallin subunits from the four fractions were characterized by one‐dimensional and two‐dimensional gel electrophoretic techniques. From the results a classification could be derived and a nomenclature for the soluble rat lens proteins is proposed. The products synthesized by rat lens mRNAs in a heterologous cell‐free system have also been characterized. Co‐electrophoresis of the radioactive products synthesized de novo together with the isolated unlabeled protein fractions on two‐dimensional gels shows the relation between primary gene products and their posttranslationally modified derivatives.
Oxidation of Carbonyl
Authors: LANIR Amos , CAUGHEY Winslow S. , CHARACHE Samuel
Published: July 2010

Oxidation reactions of carbonyl species of hemoglobins A, Zurich, and Osler with ferricyanide to form met‐hemoglobins have been investigated at pH 7.0 and 25°C. The kinetics support a two‐step process in which, first, CO is lost to give the unliganded reduced species (deoxy), followed by oxidation in a one‐electron transfer to ferricyanide ion. On and off rates for CO markedly affect the overall oxidation rate. That α subunits oxidize more rapidly than B subunits was noted by changes in C–O stretch frequency during the course of reactions with Hb A and Hb Zurich. The substitution of Arg for His at β63 in Hb Zurich resulted in a slower overall oxidation rate ascribable to greater affinity for CO. MetHb formation from stripped HbCO A was biphasic with the rate for the final ∼ 50% of the reaction about 30% faster than that for the initial ∼ 50%. Addition of inositol hexakis‐phosphate increased rates several‐fold in both phases and shifted the point of change from slow to fast phase. These effects suggest that inositol hexakisphosphate binds weakly (Kd∼ 30 μM) to Hb4(CO)4 in a high‐affinity state which, after loss of one CO, changes to a low‐affinity state where inositol hexakisphosphate binds more strongly (Kd < 1 μM) for the remainder of the reaction. With Hb Osler, which exhibits very low heme‐heme interaction and reacts poorly with inositol hexakisphosphate, the reaction was monophasic with a rate throughout comparable to the initial rate for stripped HbCO A. The very low level of CO saturation (< 1–6%) usually found in vivo with normal hemoglobin subunits should not greatly affect overall rates of methemoglobin formation from one‐electron electron acceptors. However, with the high levels of CO found bound (20–100% saturation) to the β subunit of hemoglobin Zurich in blood, the exposure to CO (as in smoking) can diminish the extent to which the abnormal Zurich subunit will be oxidized. CO exposure may, therefore, reduce significantly some of the undesired consequences of having hemoglobin Zurich in red blood cells.
Complete Assignment of the 1H NMR Spectrum of the Aromatic Residues of Lysozyme
Authors: REDFIELD Christina , POULSEN Flemming M. , DOBSON Christopher M.
Published: July 2010

Assignment of the nuclear magnetic resonance (NMR) spectrum of the 59 non‐exchangeable protons of the 13 aromatic amino acid residues of lysozyme has been completed using a combination of selective spin decoupling and nuclear Overhauser experiments. The experimental chemical shift data were used to simulate the aromatic region of the NMR spectrum at 300 MHz and at 470 MHz. Excellent agreement with the experimental spectra was obtained and the simulations permitted more accurate chemical shift values and resonance linewidths to be obtained. The current set of assignments is compared with those of the assignments made previously. Evidence for the motional behaviour of the aromatic amino acid side chains is presented.
Protein Synthesis by Isolated Acetabularia Chloroplasts
Authors: GREEN Beverley R.
Published: July 2010

Isolated chloroplasts of Acetabularia incorporate radioactive amino acids into more than 30 polypeptides in the light, including the apoprotein of the P700‐chlorophyll a protein complex, the reaction centre core of photo‐system I [Biochim. Biophys. Acta, 609, 107–120 (1980)]. In this paper it is shown that the apoproteins of the two minor chlorophyll a complexes, thought to be part of photosystem II reaction centre core, are also synthesized by isolated chloroplasts. Furthermore, they are integrated correctly into the thylakoid membrane in the absence of any cytoplasmic contribution, such that they can be isolated as chlorophyll‐protein complexes indistinguishable from those already in the membrane.

In contrast, the minor chlorophyll a + b complex ‘CP 29’ [Camm, E. L. and Green, B. R. (1980) Plant Physiol. 66, 428–432] and its dimers are not synthesized by isolated chloroplasts. In this they resemble the other chlorophyll a + b complex, the light‐harvesting complex (LHC). It may be significant that the LHC, which is not essential for photosynthetic activity, is under nuclear control, while the reaction centre polypeptides, cytochrome b559, and cytochrome f, are synthesized on chloroplast ribosomes.
Properties and Subcellular Localization of Acid Phosphatase Activity in Cultured Arterial Smooth Muscle Cells
Authors: LEAKE David S. , HEALD Benjamin , PETERS Timothy J.
Published: July 2010

Smooth muscle cells were cultured from pig aortas and their acid phosphatase activity toward 4‐methylumbelliferyl phosphate was studied. Cell homogenates hydrolysed this substrate with a pH optimum of 5.0, with very little activity at alkaline pH. The apparent Km of the activity was approximately 20 μM at pH 5.0, and the activity was directly proportional to both the incubation time and to the concentration of homogenate. Acid phosphatase activity was inhibited by p‐chloromercuribenzoic acid, by fluoride ions, and by chloroquine.

Postnuclear supernatants of the cells were subjected to analytical subcellular fractionation on sucrose density gradients. Acid phosphatase activity had a modal equilibrium density of 1.13 g/ml, which was similar to that of 5′‐nucleotidase and galactosyltransferase, marker enzymes for the plasma membrane and Golgi complex, respectively. The distribution of acid phosphatase activity also had a shoulder of equilibrium density about 1.16 g/ml, which may have corresponded to activity in the lysosomes. The activity of N‐acetyl‐β‐glucosaminidase, a lysosomal marker enzyme, had a very broad distribution with much of its activity equilibrating at low densities.

When the cells were homogenized in sucrose medium containing digitonin, a selective membrane perturbant, the N‐acetyl‐β‐glucosaminidase activity remained almost entirely in the sample layer, showing that the lysosomes had been disrupted. The modal equilibrium density of 5′‐nucleotidase activity was increased to 1.19 g/ml, whereas the modal equilibrium density of galactosyltransferase activity was not significantly affected. Acid phosphatase activity showed a similar increase in modal equilibrium density to 5′‐nucleotidase, but had a shoulder on the least dense side of its peak, which may have corresponded to activity in the Golgi complex. To confirm the plasma membrane localization of a large proportion of the acid phosphatase activity, intact cells were treated with digitonin and were then washed and homogenized in sucrose medium without digitonin. The modal equilibrium densities of both 5′‐nucleotidase and acid phosphatase activity were increased to similar extents, whereas the equilibrium densities of the marker enzymes for the other organelles were not significantly affected.

Thus, although acid phosphatase is a widely used marker enzyme for the lysosomes in subcellular fractionation studies, its activity towards 4‐methylumbelliferyl phosphate cannot be used as a marker for these organelles in pig arterial smooth muscle cells in culture.
Regulation of Rat Liver L‐Type Pyruvate Kinase mRNA by Insulin and by Fructose
Authors: NOGUCHI Tamio , INOUE Hiroyasu , TANAKA Takehiko
Published: July 2010

The effects have been studied of streptozotocin‐induced diabetes and subsequent insulin administration or feeding of a high fructose diet on the amount of enzyme protein and mRNA activity of L‐type pyruvate kinase in rat liver. Diabetes markedly decreased the L‐type enzyme activity in rat liver and insulin treatment resulted in restoration of the enzyme activity to normal. A high fructose diet also increased the enzyme activity in diabetic rats but to a lesser extent. Immunochemical analysis showed that these alterations in the enzyme activity were due to changes in the amount of immunoreactive enzyme protein. The mechanism of the changes was studied further by assaying the level of functional mRNA coding for this enzyme in a nuclease‐treated reticulocyte lysate system with total RNA isolated from rat liver. L‐type pyruvate kinase mRNA, expressed as a percentage of the total protein synthesized, was greatly decreased in diabetic rats. Insulin administration resulted in recovery of the mRNA activity to the normal level within 24 h. The lag period before accumulation of translatable mRNA of the L‐type enzyme was about 4‐5 h. The mRNA activity was also increased in diabetic rats fed a high fructose diet. This fructose effect, which was much smaller than the insulin effect, was maximal after feeding fructose diet for one day. These changes were approximately comparable to the changes in enzyme activity. Thus, it is suggested that regulations of rat liver L‐type pyruvate kinase by insulin and by fructose are primarily due to changes in the level of translatable mRNA of this enzyme.
Increase of Fidelity of Polypeptide Synthesis by Spermidine in Eukaryotic Cell‐Free Systems
Authors: IGARASHI Kazuei , HASHIMOTO Sanae , MIYAKE Atsuko , KASHIWAGI Keiko , HIROSE Seiyu
Published: July 2010

The mechanism of spermidine‐induced increase of fidelity of polypeptide synthesis in a wheat germ cell‐free system has been studied. It was found that the increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl‐tRNA to ribosomes, that reduction of misreading was more marked at the 5′‐base than at the 3′‐base of the codon and that misreading caused by paromomycin and kanamycin C was not significantly decreased by spermidine. It was deduced from these results that spermidine inhibited low‐frequency misreading more strongly than high‐frequency misreading. In addition, spermidine was found to stimulate the rejection of non‐cognate aminoacyl‐tRNA mainly at an initial discrimination step during the binding of aminoacyl‐tRNA to ribosomes, and slightly at a subsequent GTP‐dependent discrimination step, the so‐called proofreading step.

In yeast, rabbit reticulocyte, andArtemia salinacell‐free systems, spermidine was found to increase the fidelity of protein synthesis.
Enhancement of Intracellular 5‐Phosphoribosyl 1‐Pyrophosphate Levels as a Major Factor in the 6‐Azauridine‐Induced Stimulation of Carbamoyl Phosphate Synthesis in Mouse Spleen Slices
Authors: TATIBANA Masamiti , KITA Kazuko , ASAI Takashi , IKEDA Fumiaki
Published: July 2010

Brief exposure to 6‐azauridine stimulates the production of carbamoyl phosphate for de novo pyrimidine biosynthesis in vitro in slices of haematopoietic spleen from anaemic mice (preceding paper). In studies of the underlying mechanism for this response we turned our attention to changes in the level of substrates and effectors for carbamoyl‐phosphate synthetase II. Intermediates of the orotic acid pathway and 6‐azauridine had little effect on the synthetase activity in vitro. 6‐Azauridine 5′‐monophosphate (6‐AzaUMP) stimulated synthetase II, possibly in an allosteric manner. However, in view of the potency as an activator and the tissue levels, 6‐azaUMP may be only partially responsible for the stimulation. Adenine nucleotide levels in the tissue showed only minor changes after brief exposure (15 min) to 6‐azauridine. The level of UTP and UDP, potent inhibitors for synthetase II, showed no significant change. The level of 5‐phosphoribosyl 1‐pyrophosphate (PPRibP), a potent positive effector for the synthetase II, showed a more than 1.5‐fold increase after 15 min. The relative importance of these factors was evaluated by assay of the synthetase, partially purified from mouse spleen, under simulated conditions in vitro. The results indicated that the enhanced level of PPRibP played a major role in increasing the production of carbamoyl phosphate. In Ehrlich ascites cells in vitro, where 6‐azauridine did not increase carbamoyl phosphate production, the basal PPRibP level was high (range over 0.1 mM) and the changes in the level, brought about by the analogue, were relatively small.
Rat Vitamin‐D‐Dependent Calcium‐Binding Proteins
Authors: THOMASSET Monique , DESPLAN Claude , PARKES Owen
Published: July 2010

mRNA extracted from rat duodenum, kidney and cerebellum was translated in a cell‐free reticulocyte lysate system in the presence of L‐[35S]methionine. Vitamin‐D‐dependent calcium‐binding proteins (D‐CaBPs) were identified by immunoprecipitation using antibodies specific to duodenal D CaBP (7500 Mr) and cerebellar D‐CaBP (28000 Mr). When duodenal mRNA was translated, the immunoprecipitated polypeptide, obtained using antibodies to duodenal D‐CaBP, comigrated with the pure small D‐CaBP. Only the addition of unlabeled small duodenal D‐CaBP prevented the immunoprecipitation of the major protein. Likewise, when mRNA extracted from the kidney and cerebellum was translated, the product immunoprecipitated by antibodies specific to large mammalian D‐CaBP was electrophoretically similar to pure 28000‐Mr protein, being displaced only by the addition of unlabeled large D‐CaBP. The yield of the duodenal D‐CaBP synthesized in the reticulocyte lysate assay was remarkably high (about 10%) compared to that of the large D‐CaBP with renal (1%) or cerebellar (0.4%) mRNA. In the absence or presence of microsomal membranes, proteins of similar molecular weight were synthesized, suggesting that the biosynthesis of both large and small D‐CaBPs do not involve the processing of leader sequences. Moreover in our experimental conditions duodenal poly(A)‐rich RNA was unable to direct the synthesis of large D‐CaBP while the mRNAs extracted from kidney and cerebellum did not code for the small D‐CaBP. Our data indicate that two distinct mRNAs, coding for small and for large vitamin‐D‐dependent CaBPs, are expressed in specific tissues of the rat.
The Complete Amino‐Acid Sequence of the Large BacteriochlorophyII‐Binding Polypeptide from Light‐Harvesting Complex II (B800—850) of Rhodopseudomonas capsulata
Authors: TADROS Monier Habib , SUTER Franz , DREWS Gerhart , ZUBER Herbert
Published: July 2010

The large bacteriochlorophyll‐a‐binding polypeptide of the light‐harvesting complex II (B800–850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16–35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C‐terminal part is also hydrophobic while the N‐terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.
The Mechanism of Binding of L‐Serine to Tryptophan Synthase from Escherichia coli
Authors: LANE Andrew N. , KIRSCHNER Kasper
Published: July 2010

The mechanism of binding of L‐serine to tryptophan synthase, which is the initial phase of the catalytic mechanism, has been studied by steady‐state and stopped‐flow kinetic techniques. The dependence of three separable rate processes on the concentration of L‐serine is compatible with four different enzyme‐substrate complexes, one of which lies on a branch in the pathway. By use of L‐serine deuterated at the α carbon, it is possible to assign the deprotonation of the external aldimine of L‐serine with pyridoxal 5′‐phosphate to the most rapid observable binding step. Measurements at two pH values show that the rate‐determining step in the synthesis of L‐tryptophan changes from release of L‐tryptophan at the optimal pH of 7.6 to the binding of L‐serine at pH 6.5. Measurements at pH 7.6 in the presence of the substrate analogue indolepropanol phosphate show that the stronger binding of L‐serine is probably due to stabilization of the catalytically competent enzyme–L‐serine complex. At pH 7.6 L‐serine is bound far more slowly to the β2 subunit than to the α2β2 complex of tryptophan synthase and retains its α carbon proton. For the β2 subunit, the rate‐determining step of tryptophan synthesis is deprotonation of bound L‐serine. The effect of bound α subunit is to increase both the rate of deprotonation and β‐elimination, shifting the rate‐limiting step to the release of L‐tryptophan.
Delta Thalassemia: a Non‐deletion Defect
Authors: TARAMELLI Roberto , GIGLIONI Barbara , COMI Paola , OTTOLENGHI Sergio , BRANCATI Carlo , TAGARELLI Antonio , POLLI Elio , GIANNI Alessandro M.
Published: July 2010

Hematological and gene mapping studies of two Southern Italian children doubly heterozygous for δo‐ thalassemia and δoβo‐thalassemia have been carried out. No HbA2 was detectable by electrophoresis of total hemoglobin in these patients. Restriction enzyme analysis of the globin gene cluster with δ, γ, ɛ and RIH cloned probes showed all the expected normal bands, in addition to the abnormal fragments related to a previously described type of δoβo‐thalassemic deletion, demonstrating an apparently intact globin gene cluster on the thalassemic chromosome. The relevance of this finding to models for coordinated expression of non α globin genes is discussed.
Purification of 5‐Aminolaevulinate Synthase from Liver Mitochondria of Chick Embryo
Authors: BORTHWICK Iain A. , SRIVASTAVA Gopesh , BROOKER John D. , MAY Brian K. , ELLIOTT William H.
Published: July 2010

5‐Aminolaevulinate synthase from chick‐embryo liver mitochondria has, for the first time, been purified to homogeneity in its native non‐degraded form by molecular sieve chromatography, chromatofocusing and affinity chromatography. The enzyme has a minimum molecular weight of 68000 as determined by sodium dodecylsulphate/polyacrylamide gel electrophoresis and a specific activity of 35000 units/mg of protein. This result conflicts with the previous report of Whiting, M. J. and Granick, G. [(1976) J. Biol. Chem 251, 1340–1346] that the chick embryo enzyme has a molecular weight of 49000. We show here that the purified form can be degraded proteolytically to a smaller form of molecular weight around 50000 while retaining full enzymatic activity. It seem evident, therefore, that the enzyme isolated by Whiting & Granick (1976) was degraded. We have further established by pulse‐labelling studies and immunoprecipitation that the enzyme isolated by our new and rapid procedure has the same minimum molecular weight as that which exists in vivo.
The Effect of an Integral Membrane Protein on Lipid Polymorphism in the Cardiolipin‐Ca2+ System
Authors: TARASCHI Theodore F. , de KRUIJFF Ben , VERKLEIJ Arie J.
Published: July 2010

The addition of Ca2+ to aqueous dispersions of cardiolipin triggers complete hexagonal (HII) phase formation at Ca2+/cardiolipin molar ratios ≥ 1.0 as detected by 31P NMR and freeze‐fracture electron microscopy. Incorporation of the integral membrane protein glycophorin prevents the bilayer → hexagonal (HII) phase transition at Ca2+/cardiolipin ratios as high as 15:1. Removal of the outwardly oriented, negatively charged sialic‐acid‐containing sugar groups of glycophorin with trypsin had little effect on the bilayer‐stabilizing capacity of the protein. As the Ca2+ binding was found to be similar in both the cardiolipin and the cardiolipin‐glycophorin systems, it can be concluded that the protein exerts a bilayer‐stabilizing effect on the cardiolipin. In addition, the possibility that glycophorin may prevent vesicle fusion is also discussed.
Increased Urinary Excretion of Formiminoglutamic Acid in Nitrous‐Oxide‐Treated Rats and Its Reduction by Methionine
Authors: DEACON Rosemary , PERRY Janet , LUMB Michael , CHANARIN Israel
Published: July 2010

Inhalation of nitrous oxide oxidises cob(I)alamin and inactivates methionine synthetase of which cobalamin is a co‐enzyme. The biochemical changes in the rat following exposure to nitrous oxide resemble in some detail the changes present in patients with untreated pernicious anemia due to deficiency of cobalamin. There is a marked increase in the excretion of formiminoglutamic acid in the urine following exposure to nitrous oxide. A significant decrease is produced, while on N2O, by giving methionine. The explanation for these findings is discussed in the light of recent data on the effects of cobalamin inactivation.
Specificities and Selectivities of Glycerol‐3‐Phosphate Acyltransferase and Monoacylglycerol‐3‐Phosphate Acyltransferase from Pea and Spinach Chloroplasts
Authors: FRENTZEN Margrit , HEINZ Ernst , McKEON Thomas A. , STUMPF Paul K.
Published: July 2010

In addition to acyl‐CoA, purified glycerol‐3‐phosphate acyltransferases from pea and spinach chloroplasts can also use acyl‐(acyl‐carrier protein), acyl‐ACP, as a substrate for glycerol 3‐phosphate acylation. The enzyme fractions showed absolute specificity for glycerol 3‐phosphate as acyl acceptor. Dihydroxyacetone phosphate was ineffective. Glycerol 3‐phosphate was almost exclusively acylated at the C‐1 position. If mixtures of palmitoyl‐ACP, stearoyl‐ACP and oleoyl‐ACP were offered, the oleoyl group was preferred. These data fully agree with previous experiments on these enzymes carried out with various acyl‐CoA thioesters. Kinetic data determined with different acyl‐ACPs as substrates are consistent with the observed fatty acid selectivity for the oleoyl group. Double labelling experiments with mixtures of oleoyl‐ACP and oleoyl‐CoA demonstrated a preference for ACP‐thioesters.

Monoacylglycerol‐3‐phosphate acyltransferase, localized in the envelope of chloroplasts, can also utilize acyl‐ACP as substrate. Envelope fractions of spinach as well as of pea showed a high specificity for the palmitoyl group when ACP‐thioesters or CoA‐thioesters were offered and directed this acyl group to the C‐2 position of the glycerol backbone. Results from competition experiments with [14C]palmitoyl‐ACP and [3H]palmitoyl‐CoA indicate that the membrane‐bound acyltransferase preferably uses ACP‐thioesters for the acylation of 1‐acylglycerol 3‐phosphate. According to fatty acid selectivities and specificities the main product of the recombined acyltransferase systems in chloroplasts of 16:3 plants as well as 18:3 plants is a phosphatidic acid with the oleoyl group at C‐1 and the palmitoyl group at C‐2 whereas molecules with the oleoyl group at both positions are not synthesized.

Under appropriate conditions the phosphatidic acid formed by the soluble acyltransferase and spinach envelope was rapidly converted to monogalactosyl diacylglycerol in the presence of UDP‐galactose. In analogous assays with acyltransferase and envelope from pea only a low proportion of labelled phosphatidic acid was converted via diacylglycerol to monogalactosyl diacylglycerol. In both systems monogalactosyl diacylglycerol synthesized in the presence of C16:0‐thioesters and C18:1‐thioesters carried C18:1 at C‐1 and C16:0 at C‐2 in agreement with the fatty acid selectivity of the acyltransferase systems.
Characterisation of the Spore Coat Proteins of Dictyostelium discoideum
Authors: WILKINSON David G. , HAMES B. David
Published: July 2010

Using a rapid purification protocol designed to minimise proteolysis in vivo and in vitro, combined with high‐resolution one‐dimensional and two‐dimensional polyacrylamide gel electrophoresis, 11 spore coat proteins have been identified in Dictyostelium discoideum AX2 spores with apparent molecular weights of 170000 (SP170), 103000 (SP103), 97000 (SP97), 90000 (SP90), 82000 (SP82), 76000 (SP76), 72000 (SP72), 55000 (SP55), 39000 (SP39), 33000 (SP33) and 3400 (SP3). Control experiments rule out the possibility that any of these components are generated by crosslinking or proteolytic degradation artifacts. All of these spore coat proteins are disulphide‐crosslinked in the spore coat except for SP103, the majority of which is extracted in the absence of thiol reagent. All of the spore coat proteins are single polypeptides as judged by two‐dimensional polyacrylamide gel electrophoresis except for SP33 which is consistently composed of four isoelectric variants. SP170, SP103, SP97, SP90, SP82 and SP72 are extremely acidic with pl values in the range pH 4.5–4.7, SP76 and SP55 are slightly less acidic with pl values of 5.7 and 5.9 respectively, whilst the four SP33 polypeptides have pl values of pH 6.6–7.5. SP170, SP103, SP90, SP82 and SP76 are all glycoproteins as judged both by periodic acid/Schiff staining and radiolabelling in vivo with mannose, glucosamine and fucose.
Is Cytochrome b Really the Antimycin‐Binding Component of the Cytochrome b—c1 Complex of Yeast Mitochondria
Authors: CHEVILLOTTE‐BRIVET Paule , MEUNIER‐LEMESLE Danielle , FORGET Nicole , PAJOT Patrick
Published: July 2010

The antimycin binding sites were found to be independent of cytochrome(s) b synthesis in pseudo‐wild‐type revertants of cytochrome b mutants. These revertants, whose primary mutation is located in the introns of the cob‐box gene of mitochondrial DNA, have modified contents of cytochromes b‐562 and b‐565 and fully functional respiratory chain.

Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin.

It is proposed that the antimycin‐binding component is not cytochrome b itself, but interacts with it in such a way that an alteration of the cytochrome b structure affects the antimycin‐binding site.
Isoenzymes of cAMP‐Dependent Protein Kinase in Developing Rat Liver and in Malignant Hepatic Tissues
Authors: WITTMAACK Frank M. , WEBER Wolfgang , HILZ Helmuth
Published: July 2010

Total R proteins and total cAMP‐dependent protein kinase activity during rat liver development reach highest values per unit DNA when the organ has attained full metabolic competence. The parallel changes indicate coordinate synthesis of R and C subunits during hepatic development.

In contrast to total R2· C2, protein kinase activation and endogenous cAMP levels were highest around birth. Immunotitration with anti‐RI and anti‐RII in the presence of protein‐A–Sepharose of extracts obtained from various developmental stages and from hepatomas suggested a relation of both protein kinase I and II to the terminal differentiation of the organ rather than to cellular proliferation rates. The type‐II enzyme appears to be subject to additional regulations connected with neonatal adaptation phenomena.

A non‐enzymic analysis of the protein kinase activation status is described. It is based on the determination of the ratio of amounts: R · cAMP/total R, which showed a linear correlation with the conventional protein kinase activity ratio (–cAMP/+cAMP).
Heterogeneity of the Basic Pancreatic Inhibitor (Kunitz) in Various Bovine Organs
Authors: FIORETTI Evandro , BINOTTI Ines , BARRA Donatella , CITRO Gennaro , ASCOLI Franca , ANTONINI Eraldo
Published: July 2010

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10600–6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion‐exchange chromatography: they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7–4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross‐reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.
Interaction of a Peripheral Protein of the Erythrocyte Membrane, Band 4.1, with Phosphatidylserine‐Containing Liposomes and Erythrocyte Inside‐Out Vesicles
Authors: SATO Satoshi B. , OHNISHI Shun‐ichi
Published: July 2010

Inteactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside‐out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer‐containing membranes. the amount of binding to PtdSer‐containing liposomes was larger than that to PtdSer‐lacking liposomes. The amount of binding to inside‐out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside‐out vesicles decreased on PtdSer‐decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer‐containing vesicles but not with PtdSer‐lacking vesicles induced gelation of spectrin‐actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer‐containing vesicles to band 4.1 was larger than that from PtdSer‐lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer‐containing liposomes but not from PtdSer‐lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin‐actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.
Association of the Prolipoprotein Accumulated in the Presence of Globomycin with the Outer Membrane of Escherichia coli
Authors: INUKAI Masatoshi , INOUYE Masayori
Published: July 2010

The prolipoprotein, a secretory precursor of the outer membrane lipoprotein of Escherichia coli, is known to be accumulated in the cell envelope when cells are grown in the presence of a cyclic antibiotic, globomycin. The prolipoprotein was localized in the cytoplasmic membrane when it was separated from the outer membrane by sucrose‐density gradient centrifugation. However, when the envelope fraction was treated with sodium sarcosinate, the prolipoprotein was found almost exclusively in the sarcosinate‐insoluble outer membrane fraction. The prolipoprotein separated in the cytoplasmic membrane by sucrose‐density gradient centrifugation was soluble in sarcosinate and could not form a complex with the outer membrane once solubilized in sarcosinate. Labeling of the two lysine residues at positions 2 and 5 of the prolipoprotein with [3H]dinitrophenylfluorobenzene was enhanced 26‐fold when the cells were disrupted by sonication. On the other hand, a tryptic fragment of the ompA protein, which is known to exist in the periplasmic space, increased its susceptibility to [3H]dinitrophenylfluorobenzene only 5.3‐times upon disruption of the cell structure. These results indicate that the prolipoprotein accumulated in the presence of globomycin is translocated across the cytoplasmic membrane and interacts with the outer membrane. At the same time, it is attached to the cytoplasmic membrane with its amino‐terminal signal peptide in such a way that the amino‐terminal portion of the signal peptide containing two lysine residues is left inside the cytoplasm.
Calmodulin Stimulation of Adenylate Cyclase of Intestinal Epithelium
Authors: AMIRANOFF Brigitte M. , LABURTHE Marc C. , ROUYER‐FESSARD Christiane M. , DEMAILLE Jacques G. , ROSSELIN Gabriel E.
Published: July 2010

The present work analyzes the calmodulin stimulation of rat intestinal epithelial adenylate cyclase. It is shown that: (1) activation of intestinal adenylate cyclase by calmodulin occurs in the presence of 1 – 30 μM free Ca2+; (2) in the 0.5 – 10 mM MgCl2concentration range, the pattern of calmodulin action is not modified by Mg2+; calmodulin acts by increasing the maximal velocity of the intestinal adenylate cyclase without altering its affinity for ATP and Mg2+; (3) the calmodulin effect does not require the presence of guanine nucleotide and is even additive to that induced by GTP or its synthetic non‐hydrolyzable analog guanosine 5′‐[β,γ‐imido]triphosphate; (4) calmodulin and sodium fluoride exert an additive effect on the enzyme activity; (5) calmodulin and prostaglandins E1or E2display additive stimulatory actions, whatever the dose of prostaglandins tested; calmodulin, therefore, does not modify the affinity of intestinal adenylate cyclase for its hormonal effectors; (6) trifluopeazine in the 25–200 μM range of concentrations (K0.5= 85 μM) suppresses the adenylate cyclase activity which has been stimulated by 5 μM calmodulin; (7) calmodulin is active in the 0.1 – 10 μM concentration range, half‐maximal stimulation is induced by 0.9 μM protein, i.e. a dose of calmodulin that is much below the calmodulin content of the intestinal epithelial cell; (8) at low (0.5 mM) and high (5 mM) Mg2‐concentration, a sixfold and threefold maximal increase of basal activities are observed, respectively.

Our data indicate that calmodulin stimulates intestinal adenylate cyclase by a mechanism that is different from the hormonal activation and suggest the presence of a specific regulatory site for calmodulin on the enzyme.
ADP‐Ribosylation in Permeable HeLa S3 Cells
Authors: HOLTLUND Jostein , KRISTENSEN Tom , ØTVOLD Anne Carine , LALAND Søren G.
Published: July 2010

ADP‐ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4–5‐times greater in metaphase cells. 17–22% was in the soluble fraction which contained material released from the cells, 16–22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP‐ribosylated proteins corresponding to 78000, 54000 and 36000 Da. In addition metaphase cells contained several other ADP‐ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32000–39000‐Da region suggesting ADP‐ribosylation of histone H1 with up to 10 residues of ADP‐ribose and in the 17000–20000‐Da region indicating ADP‐ribosylation of core histones. The pattern of ADP‐ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP‐ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP‐ribose) and oligo(ADP‐ribose). The results do not lend support to a special function of ADP‐ribosylated histones in the mitotic event while certain ADP‐ribosylated non‐histone proteins may be specific for metaphase cells.
Latent and Active Human Polymorphonuclear Leukocyte Collagenases
Authors: MACARTNEY Henry W. , TSCHESCHE Harald
Published: July 2010

Latent and active human polymorphonuclear leukocyte collagenase could be purified to apparent homogenei by a rapid and reproducible method involving affinity chromatography on concanavalin‐A–Sepharose, collage Sepharose and activated thiol‐Sepharose 4‐B, gel filtration on Sephacryl S‐300 and ion‐exchange chromatograpl on DEAE‐Sephacel.

The latent and active collagenase were shown to be single entities with apparent Mr 91000 and 6400 respectively, as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without mercapto ethanol. Dodecyl sulfate gel electrophoresis of the latent enzyme in the presence of mercaptoethanol showed two bands with apparent Mr of 64000 and 24000 indicating the cleavage of a disulfide bond.

Activation of the native latent collagenase could be achieved by_ disulfide‐containing compounds. A concomitant decrease in molecular weight of approximately 25000 was observed on activation of the latent enzyme. The activators proved ineffective after alkylation of the latent enzyme with iodoacetamide. This lends support to the proposed thiol/disulfide interchange mechanism in the activation reaction [Macartney, H. W. and Tschesche, H (1980) FEBS Lett. 119, 327–332]. The active collagenase could be completely inhibited by an inhibitor dissociated from the latent enzyme, i.e. a complex of enzyme and inhibitor. Regulation of the collagenase activity could be achieved by the redox potential of the glutathione cycle [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190].

The latent and active enzymes were shown to be acidic proteins with isoelectric points at pH 5.5 and 6.4 respectively. Amino acid analyses of both enzymes have been obtained and indicate a difference of about 222–223 amino acid residues between the latent and active collagenases. This corresponds to a difference of approximately Mr25000 between the active and the latent enzyme, the latter being a mixed disulfide of the enzyme and its inhibitor.

The optimum pH for collagenolytic activity was observed near neutrality. The enzyme specificity was revealed to be that of a true mammalian collagenase splitting the native type I tropocollagen molecule into three‐quarter and one‐quarter fragments according to dodecyl sulfate/polyacrylamide gel electrophoresis. No other contaminating proteolytic activity was observed in the purified latent and active collagenase preparations. The active enzyme was found to be stable for over one year when stored at ‐ 70°C. The collagenase was capable of reducing the specific viscosity of a native type I collagen solution by approximately 40%.

The amount of latent collagenase present in the leukocytes was estimated to 0.65 mg/1010 cells. The overall yield obtained after the purification to homogeneity was 75–80% of the theoretical value.
The Collagenase Inhibitor from Human Polymorphonuclear Leukocytes
Authors: MACARTNEY Henry W. , TSCHESCHE Harald
Published: July 2010

A new collagenase inhibitor of latent human polymorphonuclear collagenase, initially reported by Macartney and Tschesche [Hoppe‐Seyler's Z. Physiol. Chem. 361, 298–299 (1980) and FEBS Lett. 119, 327–332 (1980)], has been purified to apparent homogeneity. We described several methods of purification of this inhibitor, but the fastest and most reproducible method is by passage of purified latent human polymorphonuclear leukocyte collagenase on activated thiol‐Sepharose 4‐B (preceding paper in this journal). Active collagenase is eluted from the column and the collagenase inhibitor is retarded. Elution of the inhibitor is achieved by eluting the column with thiol compounds in the equilibration buffer. Further purification of the inhibitor is obtained by gel filtration on Sephacryl S‐300 and ion‐exchange chromatography on DEAE‐Sephacel.

The inhibitor exhibits a strict specificity for mammalian collagenases. It inhibits collagenases isolated from human synovial fluid, human normal and diabetic skin fibroblasts and human embryonic skin fibroblasts. The inactive complexes are all subject to activation by several disulfide compounds, such as oxidised glutathione by the thiol/disulfide interchange reaction already described.

The purified inhibitor was shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (10% gel) to be homogeneous and displayed an apparent molecular weight of 24500. Titration of the inhibitor with Ellmans reagent revealed that it contains one free sulfhydryl group, a prerequisite for its inhibitory activity. The inhibitor inhibits human polymorphonuclear leukocyte collagenase in a strict 1:1 stoichiometric reaction. The amino acid composition of the complex formed from the inhibitor and active enzyme reproduces that of the latent collagenase thus confirming that the latent enzyme is a 1:1 complex.

The inhibitor is an acidic protein having an isoelectric point at pH 5.5, which would be expected from the amino acid analysis. The composition reveals a glycoprotein of about 240 residues containing a carbohydrate moiety composed of N‐acetylglucosamine, mannose, galactose and glucose. The amino acid composition of the inhibitor is not identical to that of β1‐anticollagenase from human plasma.
Characterisation of β1‐Anticollagenase from Human Plasma and Its Reaction with Polymorphonuclear Leukocyte Collagenase by Disulfide/Thiol Interchange
Authors: MACARTNEY Henry W. , TSCHESCHE Harald
Published: July 2010

A β1‐serum component, β1‐anticollagenase, capable of inhibiting various mammalian tissue collagenases, was isolated from human plasma by gel filtration, affinity chromatography and ion‐exchange chromatography. The inhibitor contains 1–2 free sulfhydryl groups, which are a preequiste for inhibitory activity and for binding to the thiol‐Sepharose affinity support. Alkylation of β1‐anticollagenase by iodoacetamide blocks inhibitory activity. The inhibitor was purified to apparent homogeneity and exhibited a Mr= 30500 determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The amino acid and carbohydrate composition was determined. According to its composition and the isoelectric focussing β1‐anticollagenase is an acidic protein with an isoelectric point of 5.6.

Inhibition of human leukocyte collagenase proceeds in a strong 1:1 stoichiometric reaction. The mechanism of this association takes place by a disulfide/thiol interchange reaction as has been previously indicated for human leukocyte collagenases in forming the latent enzyme [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327–332]. The β1‐anticollagenase‐leukocyte‐collagenase complex (latent enzyme) is activatable by disulfide‐containing compounds such as cystine, oxidised glutathione, insulin, relaxin, trypsinogen and others, but not by 179,203‐di(S‐carboxymethyl)trypsinogen, or its trypsin derivative. Compounds containing inaccessible disulfide bonds, e.g. chymotrypsin, or sulfhydryl groups, e.g. d‐penicillamine, do not activate the complex. Activation is, however, easily obtained with the oxidised‐glutathione‐generating system myeloperoxidase/H2O2/glutathione as was previously demonstrated for the human leukocyte latent collagenase activatable in a phagocytosis‐simulated respiratory burst [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190.]