The effect of growth state on the activity of the protein kinase isoenzymes an increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis, the gels were incubated with histome and [γ-³²P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 μg per channel and incorporation of ³²P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cyctosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G₁ phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G₁ and a decrease in II Type activity appears to be an early event in permitting cells to leave G₁ and resume growth.     

Disruption of the allosteric phosphorylase a regulation of the hepatic glycogen-targeted protein phosphatase 1 improves glucose tolerance in vivo

Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1–G_L) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1–G_L complex, suppressing glycogen synthesis. Consequently, the interaction of G_L with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G_L–phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G_L protein. The resulting G_L^Y284F/Y284F mice display hepatic PP1–G_L activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G_L by phosphorylase a operates in vivo. G_L^Y284F/Y284F and G_L^Y284F/+ mice display improved glucose tolerance compared with G_L^+/+ littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G_L–phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G_L^Y284F/Y284F mice lose more body weight and display decreased blood glucose levels in comparison with their G_L^+/+ littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G_L may prevent futile glucose–glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1–G_L by phosphorylase a in mammals.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.     

Purification of the murine interleukin 3 receptor.

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.     

Investigations to optimize the catalytic performance of CPO encapsulated in PEG 200-doped silica matrices

A systematic investigation of the experimental conditions that affect the performance of chloroperoxidase (CPO) when encapsulated in organic/inorganic hybrid materials was carried out, aimed at optimizing the enzymatic catalytic efficiency. Sol–gel process was used to synthesize silica matrices and the incorporation of polyethylene glycols (PEGs) has allowed us to modify the properties of the matrices and the interactions between the silica network and the enzyme. Sol–gel process conditions, i.e. PEG/TMOS (tetramethylorthosilicate) molar ratio, aging and drying time, along with the H₂O₂ concentration in the reaction mixture were optimized to obtain a more efficient and reusable catalyst. A stable and easily recycled biocatalyst was obtained, even in the presence of high amounts of oxidizing agent. A stability of up to 3 complete cycles of reaction was obtained. CPO also exhibited an excellent thermostability even at 70 °C, being residual activity after 2 h of incubation greater than 90%, and it was a very favorable result, especially in view of synthetic applications of CPO.Moreover, it was found that the immobilized catalyst performance can be maintained unchanged over at least a month simply by storing the washed matrices at 4 °C. Further optimization of the experimental conditions will lead in the future to a larger-scale synthetic use of CPO.     

Immobilization of extracellular glucose oxidase from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides

Different methods of immobilization of extracellular glucose oxidase (GO) from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides have been compared. GO from the culture liquid filtrate (CLF) associated with Zn(OH)₂ but not Al(OH)₃ gels. Preparation of samples of immobilized GO does not require isolation of the enzyme (CLF may be used). GO immobilized on Zn(OH)₂ gels from CLF was 1.6 times more efficient in catalyzing D-glucose oxidation than the enzyme contained in the original culture liquid. Crosslinking of gel-adsorbed CLF proteins affected the properties of GO adversely and to a considerable extent. Various means of polymerization and immobilization of GO isolated from CLF have been studied. Optimum results were obtained when GO polymeric products were pre-synthesized in solution, followed by adsorption to Al(OH)₃, but not Zn(OH)₂ gels. The catalytic efficiency of GO immobilized on a Zn(OH)₂ gel was significantly lower than that of the enzyme associated with Al(OH)₃.     

Structural and Biochemical Characterization of Isolated Trichocysts of the Ciliate Pseudomicrothorax dubius1

ABSTRACT Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 57% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ?20 protein bands. The major bands are at 31 and 30 kD (G₁), 27 and 26.5 kD (G₂), 25 kD, 23 kD, and six bands at 15–20 kD (G₃). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca²⁺ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G₁ and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G₃ proteins. On two-dimensional gels of trichocysts, ?40 acidic protein spots are resolved with pI's of 4.6–6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G₃, in two spots at 23 kD, in all five spots of G₁, and in seven spots over 35 kD.     

SDS-induced Peptide Conformational Changes: From Triglycyl-glycine to Amyloid-β Oligomers Associated with Alzheimer’s Disease

Sodium dodecyl sulfate (SDS) was introduced in polyacrylamide gel electrophoresis (PAGE) due to its capacity of helping proteins to become fully denatured and dissociated from each other. Numerous studies which have been undertaken, using electrospray ionization mass spectrometry (ESI–MS), reported on the process of peptide oligomerization. Many of these investigations have included tetraglycine (H₂N–Gly–Gly–Gly–Gly–COOH; G₄) as model peptide. The aim of this research is to investigate the effect of SDS on G₄ oligomerization, and, especially, to emphasize the dismantling of oligomers under micellar conditions. In water, G₄ peptide develops dimers and oligomers, which can also be evidenced in high proportion by MS in the gas phase. Although our results show that SDS is able to reduce the proportion of G₄ oligomers, the aqueous G₄-SDS system may contain G₄ dimers, G₄-SDS adducts alongside with the expected monomers and some alkaline metal adducts. The mechanism by which SDS disassembled G₄ dimers, which includes sodium ion affinity toward negatively charged carboxyl and sulfonyl groups, was also discussed. Amyloid-β peptide_1–40 conformation changed considerably and, especially, the proportion of α-helical populations increased upon SDS binding in a concentration-dependent manner. Molecular dynamics studies confirmed the tendency of Aβ molecules to form α-helical conformers, as the CD and FTIR studies showed.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G₁ phase of the cell cycle. These include the predifferentiation arrest state (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at G_D, G_S and G_N states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [³⁵S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the G_S, G_D, G_D?. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

Thermal Stability of Immobilized Glucoamylase Entrapped in Polyacrylamide Gels and Bound to SP-Sephadex C–50

Glucoamylase[α-1,4: 1,6-glucan-4: 6-glucohydroease, EC 3.2.1.3] from Rhizopus niveus was entrapped in polyacrylamide gels and adsorbed onto SP-Sephadex C–50 to elucidate the thermostability mechanism of immobilized enzymes. The thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels was enhanced slightly compared with glucoamylase in free solution, and was independent of the acrylamide monomer concentration and N, N′-methylene-bis (acrylamide) content. To explain this phenomenon, the cellular structure of polyacrylamide gel was taken into consideration in addition to interactions between glucoamylase and gel, and a decrease in dielectric constant in the gel [S. Moriyama et al., Agric. Biol. Chem., 41, 1985 (1977)¹⁾]. On the other hand, immobilized glucoamylase bound to SP-Sephadex by ionic interaction showed lower stability than free glucoamylase, and much greater stability than glucoamylase in the presence of dextran sulfate, a constituent of SP-Sephadex. Thermal stabilities for the free and immobilized enzymes were also compared at the pH not in the bulk solution, but in the SP-Sephadex.     

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system

The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K _{m app} was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Cold-Set Gelation of Commercial Soy Protein Isolate: Effects of the Incorporation of Locust Bean Gum and Solid Lipid Microparticles on the Properties of Gels

This study aimed to evaluate the ability of commercial soy protein isolate (SPI) to form cold-set gels under different pHs (5–11), pre-heating temperatures (60 °C, 80 °C), CaCl₂ (0–15 mM) and SPI (5–15%, w/v) concentrations, and also select a formulation for the investigation of the effects of incorporating locust bean gum (LBG) (0–0.3%, w/v) and solid lipid microparticles (SLM) on gels rheological and microstructural properties. Gels were evaluated in terms of visual aspect, water-holding capacity, microstructure (using confocal laser scanning microscopy and cryo-scanning electronic microscopy) and rheological properties. SPI showed higher solubilities at pHs 7 (32.0%), 9 (51.6%) and 11 (100%). Self-supported gels were obtained under several conditions at alkaline pHs. At pH 7, only systems pre-heated to 80 °C with 15% (w/v) SPI and 10 or 15 mM CaCl₂ gave self-supported gels. At neutral pH, samples showed relative structural instability, which was minimized with LBG incorporation. Formulations G_SPI (pH 7, preheated to 80 °C, 15% (w/v) SPI, 10 mM CaCl₂) and G_MIX (pH 7, preheated to 80 °C, 15% (w/v) SPI, 0.2% (w/v) LBG, 15 mM CaCl₂) were selected for emulsion-filled gels (EFG) production. Power law parameters (K′, K″), calculated from frequency sweep results, revealed that non-filled G_MIX (K′: 472.1; K″: 77.6) was stronger than G_SPI (K′: 170.4; K″: 33.6). Besides, G_MIX showed microphase separation. SLM stabilized with Tween 80-Span 80 were active fillers in EFG, altering microstructures and increasing G’, G” and the Young’s modulus (1.8 to 2.1 kPa for G_SPI and 1.4 to 2.2 kPa for G_MIX).     

Effect of carbopol and polyvinylpyrrolidone on the mechanical, rheological, and release properties of bioadhesive polyethylene glycol gels

This study examined the mechanical (hardness, compressibility, adhesiveness, and cohesiveness) and rheological (zero-rate viscosity and thixotropy) properties of polyethylene glycol (PEG) gels that contain different ratios of Carbopol 934P (CP) and polyvinylpyrrolidone K90 (PVP). Mechanical properties were examined using a texture analyzer (TA-XT2), and rheological properties were examined using a rheometer (Rheomat 115A). In addition, lidocaine release from gels was evaluated using a release apparatus simulating the buccal condition. The results indicated that an increase in CP concentration significantly increased gel compressibility, hardness, and adhesiveness, factors that affect ease of gel removal from container, ease of gel application onto mucosal membrane, and gel bioadhesion. However, CP concentration was negatively correlated with gel cohesiveness, a factor representing structural reformation. In contrast, PVP concentration as negatively correlated with gel hardness and compressibility, but positively correlated with gel cohesiveness. All PEG gels exhibited pseudoplastic flow with thixotropy, indicating a general loss of consistency with increased shearing stress. Drug release T_50% was affected by the flow rate of the simulated saliva solution. A reduction in the flow rate caused a slower drug release and hence a higher T_50% value. In addition, drug release was significantly reduced as the concentrations of CP and PVP increased because of the increase in zero-rate viscosity of the gels. Response surfaces and contour plots of the dependent variables further substantiated that various combinations of CP and PVP in the PEG gels offered a wide range of mechanical, rheological, and drug-release characteristics. A combination of CP and PVP with complementary physical properties resulted in a prolonged buccal drug delivery.     

Evaluation of diatomaceous earth as a support for sol–gel immobilized lipase for transesterification

Enzymatic production of biodiesel by triglyceride transesterification is a promising alternative to chemically catalyzed biodiesel production despite the challenges involved with using enzymes. Celite^® supported lipase sol–gels were investigated as an option for solving some of the challenges associated with the use of enzymes for biodiesel production addressing such problems as activity, stability and reusability of the enzyme. Three types of Celite^® were considered (R633, R632, and R647) and compared to unsupported lipase sol–gels. Various factors were considered with regard to comparing the support materials. They included surface morphology characterized using surface area analysis and scanning electron microscopy, physical properties including adhesion of the sol–gel to the Celite^® and the protein loading on the Celite^®, and finally enzymatic properties based on the conversion of methanol to methyl oleate and the enzymatic activity of lipase. All the sol–gels showed good conversion and initial lipase activity, and all the Celite^® supports had similar sol–gel adhesion and protein loading. Sol–gel immobilized lipase supported on Celite^® R632 had an average 6-h percent conversion of approximately 60%, and an average initial lipase activity comparable to that of the unsupported sol–gel formulation.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

Inducing enantioselective crystallization with and self-assembly of star-shaped hybrid polymers prepared via “grafting to” strategy

Helical polymers present some interesting and distinctive properties, and one of the most distinguished applications of them is the chiral recognition and resolution of enantiomers. In this work, star-shaped hybrid helical poly (phenyl isocyanide) (PPI) with polyhedral oligomeric silsesquioxanes (POSS) as the core was designed and synthesized by “grafting to” strategy. The homoarm star-shaped hybrid POSS-(PPI)₈ was first obtained by the click reaction between azide-modified POSS (POSS-(N₃)₈) and alkynyl-modified PPI (PPI-Alkynyl). The hybrid POSS-(PPI)₈ was with predominated left-handed helical conformation and exhibited excellent ability in the enantioselective crystallization of racemic compounds. In the meantime, heteroarm star-shaped hybrid (PEG)₄-POSS-(PPI)₄ was prepared by the click reaction of POSS-(N₃)₈ with PPI-Alkynyl and alkynyl-modified poly (ethylene glycol) (PEG-Alkynyl). The hybrid (PEG)₄-POSS-(PPI)₄ was amphiphilic, and it could self-assemble to form spherical micelles in aqueous solutions.     

Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates

Chaperonin 10 protein from Rattus norvegicus (Rat cpn10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label ( 1 ), attached to the N^α-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule ( 1 ) was introduced specifically onto the N^α-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10+ 1 ) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10–avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS–PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label ( 1 ), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Rhizopus delemar Lipase in Microemulsion-based Organogels: Reactivity and Rate-Limiting Study

The reactivity of Rhizopus delemar lipase immobilized in sodium bis(2-ethylhexyl) sulfosuccinate microemulsion-based organogels (MBGs) was investigated as a function of the water content in the gel. Gelatin was used as the gelling component of the MBGs. The maximal reaction rate of 6.5 μM s^?1 was obtained at a W_G (molar ratio of water to AOT in the MBG phase) value of 100. In the experimental W_G conditions, the reaction proceeded under an effectiveness factor of 0.02 to 0.04 giving the reaction-limiting regime.