The lipase from Rhizomucor miehei adsorbed on polymer beads retains substantial catalytic activity even after exhaustive drying, and the use of dry box procedures to prevent entry of atmospheric water. Rates of esterification and transesterification (alcoholysis) were measured while stirred in hexane pre-dried to similar low water activity (aw). The rate of dodecyl decanoate synthesis was over 30% of that at the optimum (aw 0.55) after drying with anhydrous CuSO4 (aw less than 10(-3)) or MgO (aw less than 10(-4). Freshly reactivated molecular sieve could cause a further reduction in, but not elimination of, activity.
The photosynthetic reaction center is one of the most complicated molecular complexes. Transducing photon energy to a transmembrane electrochemical potential difference for protons, it is the direct or indirect energy source for virtually all life. We show here that it operates in a simple, battery-like manner, with a maximum potential of 0.20 V. Intriguingly this is only one fifth of the energy of the absorbed photon. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Cardiolipin-cholesterol interactions were studied by time-resolved fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene as probe. The residual anisotropy parameter, r infinity (reflecting the fatty acid chain packing), was measured in the liquid crystalline phase as a function of cholesterol addition. Two main results are reported: (i) a slight increase of the order parameter, S, computed from the r infinity value as S = (r infinity/r0)1/2, in the physiological concentration range of cholesterol; (ii) a sharp enhancement of S from a cholesterol mole fraction (X chl) of 0.20 and up to X chl of 0.80. This is in contrast to unsaturated lecithin systems for which a continuous increase of the order parameter was monitored, culminating at X chl = 0.50, the well-known maximum level of incorporation of cholesterol into lecithin model membranes.
Cloned cDNA sequences coding for specific 72 000-74 000 Mr HeLa cell heat shock proteins (HSPs) have been used as probes to examine cytoplasmic and nuclear RNA of heat shocked HeLa cells. The mRNAs for these HSPs vary widely in size. Whilst the beta-, delta- and epsilon-HSPs are encoded in mRNAs of 1.9 kb, the gamma-HSP arises from an mRNA three times that size. A search for putative nuclear precursors to these mRNAs revealed high Mr candidates at 15.8 kb which accumulate 1-2 h after the initial heat shock, thus supporting the notion that transcriptional control is important in the initial heat shock response.
A Mr 58 000 subunit of the opiate receptor has been identified using tritiated fentanyl isothiocyanate, a potent opiate alkylating reagent with specificity for the delta-opiate receptor subclass. The subunit is alkylated in the presence of dextrorphan but not levorphanol. The specifically labelled protein was retained on columns of immobilized wheat germ agglutinin and is therefore presumably a glycoprotein. Partial purification of the Mr 58 000 opiate receptor subunit from neuroblastoma X glioma NG108-15 hybrid cell membranes is described.
Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of gamma-[32P]ATP resulted in the phosphorylation of a prominent substrate of Mr approximately 100 000 (100 kDa). One-dimensional peptide maps and two-dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (Mr approximately 97 000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+-dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)-requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC50 6-16 microM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM-depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM-dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.
A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.
A monomeric protein of Mr 40,000 that modulates the polymer state of actin has been isolated from bovine brain. When added either to preformed actin filaments or to monomeric actin, prior to polymerization, the modulator reduces the low-shear viscosity of F-actin provided that Ca2+ is present. The 40 kDa protein also inhibits the rate of actin polymerization. The inhibition is fully suppressed by removal of Ca2+ and restored by subsequent readdition of Ca2+, suggesting that the Ca2+-controlled interaction of actin with the 40 kDa modulator is freely reversible.
The amino terminal sequence of the myosin light chain (Mr = 20 000) isolated from chicken gizzards was found to be (sequence in text). This sequence assignment differs from that reported by Maita et al. [(1981) European J. Biochem. 117, 417] in the order of the tryptic peptides. The revised amino acid sequence exhibits greater homology with the phosphorylation site sequences of the regulatory light chains from cardiac and skeletal muscle. Moreover it is now apparent why synthetic peptides corresponding to the previously reported sequence were very poor substrates for the myosin light chain kinase.
The 125I-labeled Mr 80000 form of neurophysin has been purified from bovine neurohypophysi. Tryptic digests of this species were analyzed, prior to or after treatment with carboxypeptidase B, by high-pressure liquid chromatography followed by isoelectric focusing and the fragments compared with those generated by a similar treatment of reference bovine 1-39 adrenocorticotropin. The ACTH peptides 22-39 and 1-8, as well as the 1-7 derivative of the latter were identified by those two independent criteria. This provides chemical evidence supporting the hypothesis  that high Mr neurophysin may contain the sequence of ACTH.
Insulin in the presence of Mn2+ and [gamma 32P]ATP promoted the phosphorylation of two proteins of Mr 95 000 and Mr 210 000 in detergent extracts of rat liver microsomes. The Mr 210 000 protein was identified as a component od the insulin receptor by immunoprecipitation. It also bound [125I]insulin specifically, was phosphorylated largely on a tyrosine residue and could not be cleaved to smaller subunits under extreme reducing conditions. The Mr 210 000 protein appears to be a component of a sub-population of liver membrane insulin receptors in which insulin-binding and insulin-stimulated tyrosine kinase phosphorylation site(s) reside in a single polypeptide chain.
1. Introduction ‘H NMR studies of proteins > 50 000 mol. wt are thwarted by the lack of resolution of individual proton signals, even at the highest magnetic fields currently available. This is due both to the enormous number of anisochronous protons present and to the strong line broadening which results from the slow tumbling of macromolecules in solution. Greatly simplified spectra result from the recently developed photo-CIDNP technique in which a revers- ible photoreaction of a dye with solvent-exposed tryptophan, tyrosine or histidine residues generates CIDNP effects for specific protons in these residues [l-3]. The technique has already been applied to resolve specific resonances in the NMR spectrum of some relatively small proteins. We now report its application to a large protein. Glyceraldehyde3- phosphate dehydrogenase (145 000 mol. wt), with its known three-dimensional structure [4,5], was chosen for this purpose. Both the rabbit-muscle and the lobster-tail enzyme were studied. 2. Experimental Rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase was isolated and purified as in . The lobster-tail enzyme was isolated and recrystallised according to . The spectra shown here were obtained with the NAD+containing enzymes; solu- tions in 2Hz0 contained -50 mg protein/ml and 25 mM phosphate. 288 Spectra were recorded on a Bruker HX-360 spec- trometer operating in the pulse Fourier transform mode. The sample was irradiated with light pulses from a Spectra Physics model 17 1 argon ion laser (multiline, 7-W). The difference method for the detection of photoCIDNP effects in biological macro- molecules has been described [ 11. As a dye 3&car- boxymethyl-lumiflavin (a gift Dr F. Mtiller, Wageningen) was used. Chemical shifts were measured relative to the internal standard sodium 2,2_dimethyl- 2-silapentaned-sulphonate (DSS) or dioxan. The notation pHX denotes uncorrected pH-meter reading in ‘Hz0 solutions. 3. Results and discussion In fig.lA the 360 MHz proton NMR spectrum of rabbit muscle glyceraldehyde-3-phosphate dehydro- genase is shown. The broad envelope seen illustrates the difficulties mentioned above. A photoCIDNP spec- trum was obtained in the presence of 3_N_carboxy- methyl-lumiflavin ,after a pulse from the argon ion laser. The difference spectrum (light minus dark) is shown in fig.lB. Comparison of this difference spectrum with the spectrum of N-acetylhistidine under the same conditions makes it likely that at least two histidine side chains contribute to the spectrum. To verify this the pH was varied by adding small amounts of concentrated DC1 to the protein solution. The titration curves constructed in this way are shown in
Using a bromobimane fluorescent label the Mr 31 000 protein band oligomycin-sensitive (OS)-ATPase from beef heart mitochondria is shown to become much intensified by 2-mercaptopropionylglycine. In the presence of 3.5 nmol/mg protein of the thiol reagent ATP-Pi exchange activity is increased by 90%. With the fluorescent crosslinking reagent dibromobimane (DB) we show that a new fluorescent peak appears between Mr 50 000 and 60 000. ATP-Pi exchange is very much decreased by DB. The results suggest that for regulation of ATP-synthetase activity sulfhydryl groups in the region of the Mr 31 000 protein(s) play an important role.
The complete cDNA-derived sequence of a type I cytokeratin (designated no. 3) from Xenopus laevis skin is described. The deduced protein has an Mr of 51,888 and consists of a glycine-rich head domain, a well-conserved alpha-helical region and a tail rich in hydroxyamino acid residues. Various cDNA clones encoding two different mRNAs were isolated that differed by short deletions/insertions and point mutations. These microheterogeneities are mainly located in a 'hypervariable region' at the C-terminal non-alpha-helical region.
Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.
The N-terminal region of the alpha integrin subunits is predicted to fold into a beta-propeller domain. Using K562 alpha4 transfectants we show that mutations at alpha4 subunit residues Gly130 and Gly190 affect the conformation of this domain causing a reduction in the recognition of alpha4 by anti-alpha4 antibodies which map to the beta-propeller. The improper alpha4 conformation also led to an altered association with the beta1 subunit, and to a lack of alpha4beta1 adhesion to VCAM-1 and CS-1/fibronectin, as well as an abolishment of anti-alpha4- and anti-beta1-dependent homotypic aggregation. The total conservation of Gly130 and Gly190 among integrin alpha subunits suggests their importance in the correct folding of their respective beta-propeller domains, and thus, in the adhesive activity of the integrins.
Ribosomal and DNA binding proteins (DNA bp II) from an extreme thermophilic bacterium, B. caldolyticus strain EP 00275, were investigated for stability and crystallization and compared to the homologous proteins from B. stearothermophilus. Two-dimensional gel electrophoresis of both types of proteins, the amino acid composition and the sequences of some of the peptides of DNA bp II revealed a close relationship between each other. The physico-chemical characteristics of DNA bp II were similar but different from homologous proteins from T. thermophilus and C. pasteurineum. From our results we conclude: B. stearothermophilus and B. caldolyticus strain EP 00275 are similar organisms with regard to their ribosomal and DNA binding proteins.
We have determined the complete cDNA sequences of fresh water rod opsin gene (fwo) and deep-sea rod opsin gene (dso) from Japanese eel Anguilla japonica. The cDNA clones of fwo and dso consisted of 1437 and 1497 nucleotides, respectively. The predicted opsins of both genes consisted of 352 amino acid residues. Southern blot and PCR analyses of genomic DNA indicated that the Japanese eel genome contains only one fwo and one dso and they are intronless. Quantitative RT-PCR analyses revealed that the expression of fwo decreases with sexual maturation while that of dso increases.
Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC-0051 (2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium. Homogentisic acid is a key intermediate in the biosynthesis of tyrosine-derived plant quinones as well as in tyrosine metabolism. The herbicide prevented the incorporation of radioactivity from [14C]tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid. The enzyme p-hydroxyphenylpyruvate dioxygenase (EC 18.104.22.168) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC-0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants by SC-0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation. The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide.
Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG-01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (beta I/beta II) and type III (alpha) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 x 10(-5) M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor-mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.
The gene encoding a protein of 27 kDa, which is specifically expressed in Vibrio cholerae of serotype Ogawa, was identified and its nucleotide sequence determined. The plasmid carrying this gene was found to convert serotype specificity from Inaba to Ogawa when introduced into the Escherichia coli DH5(pVCI112) cell which harbors a cloned 20-kilobase genomic DNA fragment of V. cholerae NIH35A3 and expresses the 01 antigen of Inaba serotype.
A selective PKC inhibitor, UCN-01, was shown to exhibit anti-tumor activity in vitro and in vivo. We investigated UCN-01 with respect to isozyme-specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKC alpha, beta and gamma, nPKC delta, epsilon and eta, and a PKC zeta. Of the PKC isozymes examined, cPKC alpha was inhibited by UCN-01 most effectively (Ki = 0.44 nM), suggesting cPKC alpha is the prime candidate for the physiological target of UCN-01. The Ki values of UCN-01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the Ki values for nPKC isozymes are about 20 nM. Moreover, the Ki value for aPKC zeta is 3.8 microM. Thus, UCN-01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKC zeta were examined and compared with those for cPKC alpha.
The potent inhibitors of protein kinase C (PKC), H7, staurosporine, and staurosporine derivatives, were examined for their inhibitory effects on novel PKC (nPKC) isozymes delta and epsilon. H7 and staurosporine, usually used as selective inhibitors of PKC, showed similar inhibitory effects on cPKC (a mixture of cPKC alpha, beta, and gamma) and nPKC delta and epsilon. The inhibitory effects of K252a, a non-selective protein kinase inhibitor, on cPKC was 3.2- and 22-fold higher than those on nPKC epsilon and delta, respectively. The staurosporine derivatives UCN-01 and UCN-01-Me also showed selective inhibition of cPKC.
Caspase-3, a member of the caspase family of cell death proteases, cleaves cytoplasmic and nuclear substrates and promotes apoptotic cell death in mammalian cells. Although yeast homologs of apoptotic genes have not been identified, some components of apoptotic pathways retain function in yeast. Here we show that the expression of caspase-3 delays cell growth in Saccharomyces cerevesiae without causing cell death. Mutation of the caspase-3 QACRG active site abolished effects on yeast growth. Co-expression of caspase inhibitors alleviated growth inhibition in yeast as did the tripeptide caspase inhibitor ZVAD-fmk. These results suggest that substrates for caspase-3 are present in S. cerevesiae and may participate in the normal cell growth and division processes.
We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3-kinase (PI3K). AM promoted re-endothelialization in vitro of wounded monolayer of HUVECs and neo-vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA- and PI3K-dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.
We examined the ability of horseradish peroxidase (HRP), an analog of human myeloperoxidase, to protect DNA against oxidative damage caused by peroxynitrite in the presence of chlorogenic acid (CGA), a naturally occurring polyphenol. Chlorogenic acid inhibits the formation of single strand breaks in supercoiled pBR322 DNA by acting as a scavenger of peroxynitrite. Horseradish peroxidase markedly enhances the extent of DNA protection by catalyzing the decomposition of peroxynitrite in the presence of CGA. Horseradish peroxidase alone does not inhibit peroxynitrite-induced DNA strand breaks, indicating that CGA is required as an electron donor to regenerate the active enzyme. The apparent second order rate constant for the HRP-mediated oxidation of CGA in the presence of peroxynitrite at pH 6.9 is 3.4 x 10(7) M(-1) s(-1). This high rate suggests that CGA and other dietary polyphenols might efficiently scavenge peroxynitrite in peroxidase-containing systems in vivo.
Azide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier-transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)-inhibited air-oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm(-1). An additional band developed at 2062.5 cm(-1) during a longer incubation. Isotope substitutions with terminally 15N-labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high-spin heme o and CuB. The band at 2062.5 cm(-1) showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu(2+)B-N=N=N structure. Partial reduction of the oxidase with beta-NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm(-1). The former band also showed clear splittings in the presence of the terminally 15N-labelled azides, indicating that reduction of low-spin heme b alters the structure of the binuclear center leading to the Fe(3+)o-N=N=N configuration.
AHN 086, an isothiocyanato derivative of Ro 5-4864 (4'-chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [3H]AHN 086 labels a approximately 30 kDa protein in the rat pineal gland determined by both SDS-polyacrylamide gel electrophoresis and gel filtration high-performance liquid chromatography of digitonin-solubilized membranes. Specific incorporation of [3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high-affinity binding sites for Ro 5-4864) or ovalbumin. These findings suggest that the approximately 30 kDa protein labeled by [3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.
Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.
Incubating either Chinese hamster ovary (CHO) cells or 3T3-L1 adipocytes with insulin increased the phosphorylation of the eIF-4E-binding protein, PHAS-I. Insulin also activated p70S6K and the Erk-1 and Erk-2 isoforms of mitogen-activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10-100 times higher than those needed to increase PHAS-I phosphorylation and p70S6K activity. Incubating cells with the inhibitor of MAP kinase kinase (MEK) activation, PD 098059, blocked the effects of low concentrations of insulin on PHAS-I and p70S6K. The effects of the inhibitor were overcome by increasing concentrations of insulin. The results indicate that insulin activates a PD 098059-sensitive kinase that is involved in the regulation of both p70S6K and PHAS-I.
A good correlation was observed between enhanced lung cancer risk and restriction fragment length polymorphisms (RFLPs) of the P450IA1 gene with the restriction enzyme MspI. Genotype frequencies of 0.49 for the predominant homozygote, 0.40 for the heterozygote, and 0.11 for the homozygous rare allele were observed in a healthy population. Among lung cancer patients, the frequency of homozygous rare allele of P450IA1 gene was found to be about 3-fold higher than that among healthy population, and this difference was statistically significant. This is the first report to identify the genetically high risk individuals to lung cancer at the gene level.