γ-Glutamylamine cyclotransferase

Summary -Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of -(L--glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L--glutamylamines. Among its substrates are both the mono- and di--glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of -(L--glutamyl)-L-lysine in which both the -amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most -glutamyl--amino acids, nor is it active toward the -lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of -(L--glutamyl)-L-lysine in which the -amino or the -carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of -glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which -glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.     

Detection of ε(γ-glutamyl) lysine

Summary Detection of (-glutamyl) lysine crosslinks is not only necessary for establishing the importance of the dipeptide as a post-translational modification of proteins, but provides information as to the importance of the transglutaminase enzyme in a biological system. The crosslink may be detected using both indirect and direct methodology. Indirect methods for its detection include measurement of masked lysines within a protein, detection of polymer formation by gel-electrophoresis and the inhibition of crosslinking by the incorporation of small molecular weight amines into the substrate protein. Direct methods for the detection of (-glutamyl) lysine require the actual isolation of the dipeptide following its release from the sample protein by exhaustive proteolytic digestion. Separation of the dipeptide from other components of the digest may be achieved by either ion-exchange chromatography or gel filtration and its qualitative identification achieved by techniques such as paper-electrophoresis or thin layer chromatography. Quantitative estimation of (-glutamyl) lysine normally involves its further separation by ion-exchange chromatography and its post-column detection following derivatisation with ninhydrin. More recent techniques include pre-column derivatisation of the dipeptide with fluorogenic reagents such as -pthalaldehyde and separation by reverse phase HPLC. With the recent advances in liquid chromatography resulting in the improved resolution of amino acids, increased sensitivity, rapid analysis times, and small sample sizes, it appears likely that direct quantitation of (-glutamyl) lysine will be the preferred method for the future.     

ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

ATP-dependent activation of phospholipase C by antigen,NECA, Na3VO4, and GTP-γ-S in permeabilized RBL cell ghosts: Differential augmentation by ATP,phosphoenolpyruvate and phosphocreatine

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with -toxin fromS. aureus are a simplified system for the study of FcRI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP--S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to FcRI and GTP--S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na₃VO₄) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP--S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na₃VO₄ is augmented by PEP and PCr in a fashion similar to that observed for FcRI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by FcRI or Na₃VO₄. Together, these experiments demonstrate that antigen, Na₃VO₄, GTP--S, and NECA can all activate PLC in permeabilized RBL cell ghosts in the presence of ATP and that these activities are differentially affected by the addition of a second compound with an energetic phosphate bond.Abbreviations DNP₂₅BSA bovine serum albumin derivitized with 25 dinitrophenyl groups per mole of BSA - FcRI high affinity receptor for IgE - GTP--S guanosine-5-O-(3-thiotriphosphate) - IPs inositol phosphate(s) - K₂ Pipes dipotassium Pipes - Na₃VO₄ sodium vanadate - NECA 5-(N-Ethyl)carboxamidoadenosine - PCr phosphocreatine - PEP phosphoenolpyruvate - PLC phospholipase C specific for phosphoinositides - RBL rat basophilic leukemia     

Brightness generation in the human visual system

In this study we show that it is possible to derive experimentally measured Colour-Brightness Signals from 3 fundamental sensitivity functions. The cortical Colour-Brightness Signal is computed from the weighted summation of all afferent colour difference signals, cortically decoded. Large retinal bipolar signals are limited by ganglion cell nonlinearities. To account for colour adaptation, we use as a model principle a signal-adaptative gain regulation of the receptors. A logarithmic transformation of receptor signals is assumed. The summation of blue receptor signals which varies as a function of the adaptation level is described by a multiplication factor where <1.     

Phylogenetic Relationships of the Seven Coat Protein Subunits of the Coatomer Complex, and Comparative Sequence Analysis of Murine Xenin and Proxenin

The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated , , , , , , and COP. Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution. Both nucleotide and protein trees displayed close relationships between and subunits, between and subunits, and between and subunits, implying evolution from common ancestors as well as functional similarity. Interestingly, although 6 out of 7 -COP genes appeared to be grouped and related to the -COP genes, 4 out of 7 -COP gene products clustered with other groups of other COP subunit proteins. A 5 coding segment of the murine -COP gene was amplified by RT-PCR and cycle-sequenced. The partial predicted amino acid sequence of this murine homolog was exactly identical to the human and bovine counterparts. Of particular significance was the complete identity of the first 25 and 35 N-terminal residues which constitute the gastrointestinal hormone xenin and its precursor proxenin, thus emphasizing their strict evolutionary conservation and alluding to their physiological importance.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

PKCε inhibits the hyperglycemia-induced apoptosis signal in adult rat ventricular myocytes

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC _I/_II, and ) or activate (PKC). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC _I/_II and blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKC (V1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKC translocation activator (RACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKC in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKC protects ARVM from the hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)     

The human genes for the α and γ subunits of the mast cell receptor for immunoglobulin E are located on human chromosome band 1823

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It is tetrameric complex of one subunit, one subunit, and two disulfide-linked subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to the subunit of FcRI also form cell surface complex with other Fc receptors such as mouse FcRIIa in macrophages and most probably with human FcRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for the (FCER1A) and subunits (FCER1 G) of FcERI and the gene for FcRIII (FCGR3, CD16) are located on human chromosome band 1823.     

Polymerization Sites in the D-Domain of Human Fibrin(ogen)

The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC₅₀ = 0.06 M). The inhibitory effect of the D-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains _DD (B134-461) and _DD (63-411)₂ of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of _DD and _DD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC₅₀ values for _DD and _DD were 0.24 and 0.10 M, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in B134-461 and 63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the -chain and a hypothesis about -chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

The association of the human ε-globin gene with the nuclear matrix: a reconsideration

The association of the human -globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human -globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G/A-globin gene domain, the 16 kb /-globin gene domain and the 10 kb -globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association.Summarizing our results we could say that a region starting 5.7 kb 5 to the human -globin gene and ending 4 kb 3 to the human -globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.     

Structural characterization of thermal prebiotic polypeptides

Summary Thermal polycondensation of amino-acids as a possible prebiotic path of chemical evolution of life has been critically examined.The polymeric materials studied by nmr methods have scarce resemblance to natural peptidic material because , and peptide bonds largely predominate over -peptide bonds.     

Transglutaminases and the clotting of mammalian seminal fluids

Summary Rapid coagulation of seminal fluid in rats, guinea pigs, and several other mammalian species including certain non-human primates is responsible for the post-coital formation of copulatory plugs in the vagina. The clotting of rodent seminal plasma results from coagulation of certain proteins derived from the seminal vesicles by enzymes secreted mainly by the coagulating (anterior prostate) gland. Several lines of evidence indicate that the clotting enzymes of coagulating gland secretions are trans glutaminases, and that the extreme insolubility of the seminal clot in rodents is due to transglutaminase-catalyzed formation of (-glutamyl)ly-sine cross-links between polypeptide chains. Various features of the apparently unique forms of transglutaminases produced by rat coagulating gland and the actions of these enzymes on vesicular secretory and other proteins are discussed. The aliphatic polyamines spermidine and spermine are incorporated covalently into the proteins of the clot as the corresponding N-mono--(-glutamyl)- and N,N-bis(-glutamyl)-adducts during the enzymatic coagulation process. At the greater than millimolar concentrations at which cross-spermidine and spermine are present in normal rat seminal plasma, these polyamines attenuate the formation of hard clots in reconstituted rat semen coagulation systems, seemingly by competing with lysyl residues in vesicular secretion proteins as transglutaminase amine donor substrates, and thus preventing formation of -(-glutamyl)lysine cross-bridges. It is proposed that in those species such as the rat and man in which seminal plasma contains large amounts of spermidine and(or) spermine of prostatic origin, the seminal polyamines may serve to stop blockage of the urethra by preventing too explosive a rate of seminal clot formation during the ejaculatory process.     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

A hypervariable region at the D19S11 locus

Summary The polymorphic locus D19S11 consists of four closely linked RFLPs: , , and on chromosome 19p13.219cen, revealed by subclones p13-1-82 and p13-2-21 from cosmid 1–13. Here, we report that p13-1-25, an additional subclone of c1-13, reveals three insertion/deletion RFLPs, , , and , at the D19S11 locus. In situ hybridization of p13-1-25 to metaphase chromosomes from a carrier of a 19/X translocation with a breakpoint near the centromere confirms localization of D19S11 to 19p. Studies with hydatidiform moles have generated assignments of specific restriction fragments to these three loci, and genotypic studies in three-generation families have indicated that they are closely linked. Loci (also detected by p13-1-82) and each have but two common alleles, whereas has at least 33 alleles, including a null allele. Fifty unrelated individuals tested displayed unique fragment patterns on Taq I blots probed with p13-1-25. Applications of this probe include monitoring loss of chromosome 19 during tumorigenesis, monitoring engraftment of donor bone marrow after transplantation, testing for paternity, and mapping disease genes on chromosome 19.     

The chloroplast ATP synthase: Structural changes during catalysis

This article summarizes some of the evidence for the existence of light-driven structural changes in the and subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of the subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modifyLys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the subunit; and (3) release/ exchange of tightly bound ADP from the enzyme. These and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Stabilization of collagen-tailed acetycholinesterase in muscle cells through extracellular anhorage by transglutaminase-catalysed cross-linking

A component of collagen-tailed acetylcholinesterase (asymmetric; A-AChE) in muscle forms a metabolically-stable pool which can be released from the cell surface only by collagenase, suggesting that part of the enzyme is covalently bound by its tail (COL.Q) subunits. We have investigated whether this insoluble pool forms through covalent cross-linking of A-AChE to extracellular matrix glycoproteins by tissue transglutaminase (Tg; type 2 transglutaminase). Tg catalyzed the incorporation of the polyamine substrate³[H]-putrescine into the collagen tail of affinity-purified avian A12-AChE. Complexes between A12-AChE and cellular fibronectin were also formed in vitro by Tg. In quail myotubes, retinoic acid, which stimulates the formation of (-glutamyl)lysine isodipeptide bonds by Tg in myotubes, increased the proportion of extraction-resistant (er) A-AChE. Following irreversible inactivation of AChE by diisopropylfluorophosphate, entry of newly-synthesized A-AChE into the extraction-resistant pool was inhibited by a competitive Tg inactivator RS48373-007. The quantity of exogenously-added A12 AChE incorporated into the extraction-resistant pool in living myotubes was increased by Tg in the presence of calcium. The inhibition of cross-bridge formation in fibrillar collagen by -aminopropionitrile, and pre-exposure of myotubes to a monoclonal antibody to fibronectin, resulted in a reduction in the size of the erA-AChE pool present on the cell-surface. The evidence supports the hypothesis that a component of insoluble collagen-tailed AChE, once subject to clustering influences mediated via reversible docking to proteoglycans and their receptors, is anchored at the cell surface through covalent cross-linking by Tg. The high stability of the (-glutamyl)lysine isopeptide bond is likely to contribute to the observed low turnover of the erA-AChE fraction.     

N-(indol-3-ylacetyl)amino acids as sources of auxin in plant tissue culture

N-(Indol-3-ylacetyl) derivatives (IAA conjugates) of aliphatic amino acids with a two- to six-carbon backbone including -l-amino acids, (-amino acids, and the ,-diamino acids ornithine and lysine were prepared, chemically characterized, and tested as sources of auxin in plant tissue culture. Stimulation of unorganized growth in Solanum nigrum L. callus and callus induction and developmental effects in tomato (Lycopersicon esculentum Mill. cv. Marglobe) hypocotyl explants were studied systematically. Relative auxin activities were estimated by comparing physiologically equivalent concentrations, in the optimal and suboptimal range, of the individual IAA conjugates. While the growth-promoting properties of some of the conjugates were species-dependent, those containing straight-chain two- to four-carbon -l-amino acid moieties were generally up to 100 times more active than those of their five- to six-carbon homologues. Branching of the amino acid backbone at C- (norvaline vs. valine and norleucine vs. isoleucine) and C- (norleucine vs. leucine) had a minor effect, but substitution of H- by a methyl group (-amino-l-butyric vs. -aminoisobutyric acids) almost completely blocked growth-promoting activity. IAA conjugates of -amino acids were, in most cases, nearly as active as those of their -amino-l-isomers. Among the conjugates of ,-diamino acids N -(IAA) ornithine was less active than N -(IAA)lysine. The activity of N -(IAA)lysine was less than for the -(IAA) isomer, and that of N ,N -(IAA)₂-lysine was different in tomato and Solanum nigrum. The l-alanine and -lysine conjugates were also found to be useful for induction and development of Oenothera leaf callus and in tomato cell-suspension culture, two systems which require highly active sources of auxin.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indol-3-ylacetic acid the abbreviations for N-(indol-3-ylacetyl)amino acids are listed in Table 1.