α-Factor inhibition of the rate of cell passage through the “start” step of cell division in Saccharomyces cerevisiae yeast: Estimation of the division delay per α-factor·receptor complex

A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t_1/2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, K_I, represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10⁻¹⁰M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K₅₀ (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10⁻⁸M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant K_D and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10⁻⁸M and 10⁴, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 nM. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed.     

Kinetic evidence for a unique testosterone-receptor complex in 5α-reductase sufficient genital skin fibroblasts and the effects of 5α-reductase deficiency on its formation

When 5α-reductase-sufficient genital skin fibroblast (GSF) monolayers are incubated with testosterone (T), they first form androgen (A)-receptor (R) complexes that dissociate at a fast rate [k(37°C = 0.024 min⁻¹]. As T is converted to 5α-dihydrotestosterone (DHT), this population of T-R complexes is eventually replaced by one that dissociates much more slowly [k(37°C) = 0.006 min⁻¹], at a rate typical of DHT-R complexes. During the course of T to DHT conversion, one may observe a population of A-R complexes that has a linear (monophasic) intermediate dissociation rate constant [k(37°C) = 0.012 min⁻¹]; this population cannot simply reflect a mixture of T- and DHT-R complexes. The rate at which the complexes are processed from one dissociative form to the next varies with the incubation temperature and the presence or absence of serum in the medium; it also varies within and among GSF strains under apparently constant conditions. To explain these facts, we propose a model that enables the 5α-reductase enzyme to influence the processive dissociative behaviour of T-R complexes by engaging in some sort of coupling with the AR. The proposal is strengthened by a set of observations in cells with constitutive, mendelian or inhibitor-induced 5α-reductase deficiency that preclude a simple quantitative relation between A-R complex processing and the extent of T to DHT conversion.     

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to α-factor requires the CDC25 gene product

The α-factor mating pheromone induces a transient intracellular alkalinizatin of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinotrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and ‘schmoo’ formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the α-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25^ts mutation causes MATa cells to become nonresponsive to α-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Thermostable α-amylase from derepressed Bacillus licheniformis produced in high yields from glucose

High yields of thermostable α-amylase was produced by Bacillus licheniformis 44MB82-G, resistant to glucose catabolite repression, on the basis of inexpensive raw materials and glucose as a main carbon source. The optimal parameters for the α-amylase production were an agitation rate of 500 rpm, constant air-flow rate (1 vvm) and cultivation temperature 40°C. An enzyme activity of 4800–5000 U/ml culture medium was reached in 96–120 h. The α-amylase preparation had the following characteristics: α-amylase activity 55 000 U/ml, high thermostability (98% residual α-amylase activity after 10 min treatment at 90°C), protein content 88 mg/ml and dry substances 30%.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Binding of estrogens by α-fetoprotein in rat amniotic fluid

Amniotic fluid from 15–17-day rat fetuses bound estrone and 17β-estradiol specifically. Related steroids such as estriol, 6-ketoestradiol, 17α-estradiol and testosterone were not bound to any significant extent. The apparent K_a for 17β-estradiol was 2.6·10⁸ M⁻ at 4°C; 6 nmoles of 17β-estradiol were bound per ml of amniotic fluid. The binding component appears to be α-fetoprotein in that it migrates as an α₁-globulin upon polyacrylamide gel electrophoresis and has an isoelectric pH of 4.7 as determined by isoelectric focusing. Furthermore, binding activity was precipitated by antiserum which was shown by immuno-electrophoresis to be specific for α-fetoprotein. Binding activity, partially purified by isoelectric focusing of amniotic fluid, was associated with one of two bands seen by polyacrylamide gel electrophoresis. This band migrated as an α₁-globulin.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Prostaglandin F2α, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: Effects of oxytocin, luteinizing hormone, prostaglandin F2α and estradiol-17β

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 × 10⁵ cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F₂α (PGF), oxytocin (OT), estradiol-17β (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 ± 66.2, 111.1 ± 37.8, 57.7 ± 15.4 and 124.3 ± 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P<0.01) than on Day 8, 14 and 18 (rmOT: 17.5 ± 2.6 versus 5.6 ± 0.7, 6.0 ± 1.4 and 3.1 ± 0.4 pg/ml; P: 138.9 ± 19.5 versus 23.2 ± 7.5, 35.4 ± 6.5 and 43.6 ± 8.1 ng/ml, respectively). Oxytocin increased (P<0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17β stimulated (P<0.05) PGF secretion on Days 8, 14 and LH increased (P<0.01) PGF production only on Day 14. Prostaglandin F₂α, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P<0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P<0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated. The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h.The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol.It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.     

Sequence of the 68,869 bp IncP-1α plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements

To analyse the significance of conjugative broad-host-range IncP-1α plasmids for the spread of antibiotic resistance determinants in waste-water treatment plants we isolated and characterised five different IncP-1α plasmids from bacteria of activated sludge and the final effluents of a municipal waste-water treatment plant. These plasmids mediate resistance to ampicillin, cefaclor, cefuroxime, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, tobramycin, and trimethoprim. The complete 68,869 bp DNA-sequence of the IncP-1α plasmid pTB11 was determined. The pTB11 backbone modules for replication (Rep), mating pair formation (Trb), multimer resolution (Mrs), post-segregational killing (Psk), conjugative DNA-transfer (Tra), plasmid control (Ctl), and stable maintenance and inheritance (KilA, KilE, and KilC) are highly conserved as compared to the ‘Birmingham’ IncP-1α plasmids. In contrast to the ‘Birmingham’ plasmids pTB11 carries an insert of a Tn402-derivative integrating a class 1 integron in the intergenic region between the multimer resolution operon parCBA and the post-segregational killing operon parDE. The integron comprises the resistance gene cassettes oxa2 (β-lactamase), aacA4 (aminoglycoside-6′N-acetyltransferase), and aadA1 (aminoglycoside-3′-adenylyltransferase) and a complete tniABQR transposition module. Integron-specific sequences were also identified on other IncP-1α plasmids analysed in this work. In contrast to the ‘Birmingham’ plasmids the pTB11 tetracycline resistance module carries a pecM- and a pncA-like gene downstream of the tetracycline resistance gene tetA and contains an insertion of the new insertion sequence element ISTB11. The transposable elements IS21 and Tn1 which disrupted, respectively, orf7 and klcB on the ‘Birmingham’ plasmids are not present on pTB11. Identification of IncP-1α plasmids in bacteria of the waste-water treatment plant’s final effluents indicates that bacteria carrying these kind of plasmids are released into the environment.     

Biotransformation of (±)-α-ionone and β-ionone by cultured cells of Caragana chamlagu

Suspension cultures of Caragana chamlagu (Leguminosae) convert (±)-α-ionone (1) into (±)-3-oxo-α-ionone (3) as the major product and β-ionone (2) into 5,6-epoxy-β-ionone (6) as the sole product. It is interesting to note that the cultured cells of C. chamlagu convert regioselectively the cycloolefinic part of 1 into the corresponding unsaturated carbonyl compound, allylic alcohol and epoxide as the oxidation products, whereas the suspension cultures of Nicotiana tabacum (Solanaceae) convert the unsaturated carbonyl of 1 into the corresponding saturated ketones and alcohols as reduction products.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

A study of the reactivity and structure of cyclic α,β-unsaturated Fischer-type carbene complexes

Cycloaddition reactions with α,β-unsaturated carbene complexes of the Fischer-type bearing the carbene carbon atom and the double bond incorporated in the same ring are described. Pentacarbonyl(2H-benzopyran-2- ylidene)chromium(0) complexes (2a-c) and pentacarbonyl(4-methoxy-3,3-dimethyl-2-oxacyclopentylidene)- chromium(0) (3) show a rather low reactivity towards 1,3-dipoles and 1,3-dienes. The reactions with diazomethane are regioselective but not chemoselective; compounds 2 and 3 show two sites of attack: the α,β carbon-carbon and the carbon-metal double bond. The crystal and molecular structures of 2a and 3 have been elucidated by single crystal X-ray analysis. Crystals of 2a are monoclinic, space group P2₁/c, a=7.614(3), b=14.033(3), c=12.766(3) Å, β=95.24°, V=1358.3(7) Å Z=4; crystals of 3 are triclinic, space group P , a=6.553(1), b=9.408(1), c=10.620(1) Å α=92.70(1), β=92.30(1), γ=92.12(1)°, V=653.0(1), ų, Z=2. Final agreement indices for 2a and 3 are R=0.034 and 0.033, respectively. Vibrational properties of the Cr(CO)₅ moiety were interpreted by FT-IR and FT-Raman spectroscopy. Electronic spectra and π electron distribution were interpreted by resonance Raman spectroscopy.     

Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta

Laminins assemble into trimers composed of α, β, and γ chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin α1 and α5 chains. Comparative pulse–chase experiments and deglycosylation studies in JAR cells established that the M_r 360,000 laminin α1 chain is glycosylated into a mature M_r 400,000 band while the M_r 370,000 laminin α5 chain is glycosylated into a M_r 390,000 form that upon secretion is further processed into a M_r 380,000 form. Hence, despite the shorter peptide length of α1 chain in comparison with the α5 chain, secreted α1 assumes a larger size in SDS–PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin α1 and laminin α5 chains in laminin-1 and laminin-10. In placenta laminin α1 chain (M_r 400,000) and laminin α5 chain (M_r 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin α1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin β2 chains. Surprisingly, a fraction of the laminin α1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin β1–β3 chains, possibly pointing to an unexpected complexity in the chain composition of α1-containing laminin isoforms.