Structural effects of terminal groups on nonenzymatic and enzymatic degradations of end-capped poly(L-lactide)

Poly(L-lactide) (PLLA) with various alkyl ester chain end groups were synthesized by ring-opening polymerization of L-lactide in the presence of zinc alkoxide as a catalyst. The structural effect of chain end groups on the rate of enzymatic and nonenzymatic degradations for amorphous films of PLLA were investigated at 37 degrees C in a Tris-HCl buffer solution (pH 8.6) with proteinase K and at 60 degrees C in a phosphate buffer solution (pH 7.4), respectively. The rate of enzymatic degradation for PLLA films was dependent on the carbon numbers of alkyl ester chain end groups, and the rates of PLLA samples with dodecyl (C12), tridecyl (C13), and tetracocyl (C14) ester end groups were much lower than those of the other samples. The surface morphologies of PLLA films after enzymatic degradation were characterized by scanning electron microscopy. After the enzymatic degradation, non-end-capped PLLA, PLLA with methyl (C1) and hexyl (C6) ester chain ends, were degraded homogeneously by proteinase K and the film surface was very smooth. In contrast, the PLLA with alkyl ester chain ends of carbon numbers over 12 were degraded heterogeneously by the enzyme, and the sponge-like network structure was formed on the film surface. These results indicated that the long alkyl ester groups at the chain ends of PLLA molecules aggregated in the amorphous films and the erosion rate was depressed due to the coverage of the aggregated terminal groups on the film surface. For the nonenzymatic degradation, the molecular weight of non-end-capped PLLA was remarkably decreased with progress of degradation. In contrast, the molecular weight of the end-capped PLLA gradually reduced at the initial stage of degradation and then the rate of degradation was accelerated. The decreases of molecular weight of PLLA by autocatalyzed degradation were retarded by the capping of carboxyl chain ends.     

Molecular weight effects on the glass transition of gelatin/cosolute mixtures

The structural properties of four gelatin fractions in mixture with sucrose and glucose syrup have been investigated extensively using small deformation dynamic oscillation. The total level of solids was 80%, the number average molecular weight of the protein ranged from 29.2 to 68 kD, and the temperatures were between 60 and -60 degrees C. Remarkably, the nature of the time and temperature dependence on the viscoelastic functions of all samples could be reduced to master curves using horizontal shift factors. The construction of master curves indicates a common mechanism of structure formation, which, in accordance with the synthetic polymer literature, comprises the rubbery zone, glass transition region, and glassy state. Application of Ferry's free-volume formalism and Rouse theory suggests that there is no change in the thermodynamic state of materials during vitrification, with changes in molecular weight simply introducing shifts in the time scale and temperature range of contributions to viscoelasticity. The thermorheological simplicity allowed development of the concept of "rheological" Tg. This was defined as the point between free-volume phenomena of the polymeric backbone occurring in the glass transition region and an energetic barrier to rotation required for local chain rearrangements in the glassy state. Mechanical relaxation and retardation distribution functions were calculated, thus obtaining values for the effective friction coefficient per monomer unit of the protein. It appears that the local friction coefficient is governed by a linear relationship between fractional free volume and the decreasing molecular weight of the protein, which introduces additional voids due to molecular ends.     

Characteristic chain-end racemization behavior during photolysis of poly(L-lactic acid)

Photolysis of poly(L-lactic acid) (PLLA) has many unclear points, such as the degradation mechanism, kinetics, products, and racemization mechanism. To clarify these features of PLLA photolysis, we examined the relationship between photolysis and racemization. The hexad stereosequential analysis of photodegraded PLLA was investigated to specify the racemized positions within a chain in comparison with hydrolysis and thermal degradation. Results from (13)C NMR spectra of UV-irradiated PLLA samples indicated that the samples have racemized d-lactate units at chain ends. From the comparison of racemization behavior among photolysis, hydrolysis, and thermal degradation, it was confirmed that the preferential racemization behavior of each of these three degradation processes is characteristic and distinct, being identified as chain-end racemization, poor racemization, or internal-unit racemization, respectively. The characteristic chain-end racemization behavior of photolysis was first confirmed in this study.     

Branched poly(lactide) synthesized by enzymatic polymerization: effects of molecular branches and stereochemistry on enzymatic degradation and alkaline hydrolysis

In this article the effects of the number of molecular branches (chain ends) and the stereochemistry of poly(lactide)s (PLAs) on the enzymatic degradation and alkaline hydrolysis are studied. Various linear and branched PLAs were synthesized using lipase PS (Pseudomonas fluorescens)-catalyzed ring-opening polymerization (ROP) of lactide monomers having different stereochemistries (L-lactide, D-lactide, and D,L-lactide). Five different alcohols were used as initiators for the ROP, and the monomer-to-initiator molar feed ratio was varied from 10 to 100 and 1000 for each branch in the polymer architecture. The properties of branched PLAs that would affect the enzymatic and alkaline degradations, i.e., the glass transition temperature, the melting temperature, the melting enthalpy, and the advancing contact angle, were determined. The PLA films were degraded using proteinase K or 1.0 M NaOH solution, and the weight loss and changes in the number average molecular weight (Mn) of the polymer were studied during 12 h of degradation. The results suggest that an increase in the number of molecular branches of branched PLAs enhances its enzymatic degradability and alkali hydrolyzability. Moreover, the change in Mn of the branched poly(L-lactide) (PLLA) by alkaline hydrolysis indicated that the decrease in Mn was in the first place dependent on the number of molecular branches and thereafter on the length of the molecular branch of branched PLA. The branched PLLA, poly(D-lactide) (PDLA), and poly(D,L-lactide) (PDLLA) differed in weight loss and change in Mn of the PLA segment during the enzymatic degradation. It is suggested that the branched PDLLA was degraded preferentially by proteinase K.     

Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.

We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Thermal degradation processes of end-capped poly(L-lactide)s in the presence and absence of residual zinc catalyst

Thermal degradation processes of end-capped poly(L-lactide)s (PLLAs) were investigated by means of several thermoanalytical techniques under both isothermal and nonisothermal conditions. Based on the thermogravimetric analysis, it was found that two distinct processes at temperatures below and above 330 degrees C were existed in the nonisothermal degradation for PLLA samples depending on the amounts of residual zinc compounds from synthesis process. Isothermal degradation experiments at different temperature regions showed that the sample weight of PLLA decreased linearly with time in both cases, whereas the changes in molecular weight revealed different tendency for the temperature. On the basis of characterization of the residual PLLA molecules after isothermal degradation at 330 degrees C, it was confirmed that the omega chain end of the residual molecules was an acrylic ester unit. Majority of volatile products during thermal degradation of PLLA was the lactide regardless of the degradation temperature. From these results, it is concluded that, during the thermal degradation of PLLA samples in the presence of large amounts of residual zinc compounds, the zinc compounds catalyze both the intermolecular transesterification generating PLLA with low molecular weights and the selective unzipping depolymerization of PLLA with low molecular weights at temperatures below 330 degrees C. In contrast, the primary reaction of thermal degradation for PLLA in the absence of residual zinc compounds above 330 degrees C is a competition between the random chain scission via a cis-elimination reaction and the cyclic rupture via intramolecular transesterification of PLLA molecules. In addition, it was evidenced that the racemization of lactic acid units in the main chain of PLLA molecules occurred at temperatures above 330 degrees C.     

Comparative studies of Feulgen hydrolysis for DNA. I. Influence of different fixatives and polyethylene glycols

We compared the Feulgen hydrolysis curves (37 degrees C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20 degrees C; ethanol-acetone, 1:1, 120 min at 4 degrees C; ethanol-glacial acetic acid mixture (3:1), containing 2% of formaldehyde (EAF), 90 min at 20 degrees C; and ethanol-glacial acetic acid (3:1), 60 min followed by 5% chrome alum solution for 360 min at 20 degrees C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest "plateau" region and the smallest scattering of DNA-Schiff amount values along the "plateau". With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG6000 on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest "plateau" was obtained with PEG6000. The only effect of PEG1500 was a dramatic increase of DNA-Schiff amount at the peak point. PEG20000 had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's "chain with a stable structure" model of Feulgen hydrolysis.     

Immunogenicity of hepatitis B vaccine in guinea-pigs, mice and man

The immunogenicity of the hepatitis B vaccine Hevac B Pasteur was assayed in guinea-pigs. The reproducibility of the test was determined by comparing the ImD50 of two samples of vaccine in 12 and eight groups of 40 animals each. The variation coefficient between groups was approximately 30%, similar to that found for other vaccines assayed in biological tests in vivo. The sensitivity of the test was determined by comparing the dose response curves in mice, guinea-pigs and humans. The results showed that one ImD50 in man (after one injection) was approximately equivalent to 3 ImD50 in guinea pigs and to 100 ImD50 in Balb/C mice. Stability of Hevac B at 37 degrees C was assayed by determining in guinea-pigs the ImD50 of a series of samples of vaccine that had been incubated at 37 degrees C for varying amounts of time. The immunogenicity of the vaccine was found to decay with a half-life of ten to 15 days at 37 degrees C.     

Microbial desizing using starch as model compound: enzyme properties and desizing efficiency

A film of sizing agents protects yarn during weaving. Its removal in a subsequent washing process causes 50% of the organic effluent load of textile finishing processes and requires large amounts of auxiliary chemicals (e.g., surfactants). Microbial desizing is a new bioprocess that uses the acidifying culture of a two-phase anaerobic digestion plant for the removal and partial degradation (acidification) of the sizing agent. Soluble starch is used in this study to characterize the enzymatic properties in the supernatant of the desizing culture and to link them to desizing efficiencies. The supernatant of the culture (grown at 37 degrees C, pH 5.5) displayed the highest enzymatic activity between pH 4 and 5 and in a broad temperature range (20-80 degrees C). Highest metabolization rates were determined with the substrate amylose. Short chain dextrins (average of 5 and 10 glucose units) and amylopectin were converted significantly more slowly. At 37 degrees C the half-life time of the enzymatic activity in the supernatant was 45 h. In a desizing test a decisive reduction of the chain length was found already after 1 h (allowing starch solubilization). A microbial desizing experiment with dyed, native maize starch demonstrated the efficiency of the proposed bioprocess.     

pH measurement in human skeletal muscle samples: effect of phosphagen hydrolysis

Measurements of muscle pH (pHm) with the homogenate technique are routinely made when extensive phosphagen hydrolysis has occurred. Upon exposure of the homogenate to 37 degrees C in the pH meter, phosphocreatine and ATP were rapidly degraded to 35 and 60% of control concentrations after 30 s. Attempts at chemically arresting this hydrolysis were unsuccessful. Therefore we examined the significance of phosphagen hydrolysis on pHm measurement in human biopsies taken at rest and following intense electrical stimulation. To accomplish this, pHm was measured at 0 degree C, where extensive hydrolysis did not occur. On the same homogenate, pHm was measured at 0 degree C with phosphagens and at 0 and 37 degrees C after phosphagen hydrolysis. The effect of phosphagen hydrolysis on pHm at 0 degrees C was used to estimate this effect at 37 degrees C. In resting samples, phosphagen hydrolysis produced a nonsignificant acidification of 0.008 pH units and, in electrically stimulated samples, a nonsignificant alkalinization of 0.033 units. Measurements of homogenate PCO2 suggested that most of the CO2 remained in the sample during pHm measurement at 37 degrees C. The present work substantiates the use of the homogenate technique as an accurate and practical method for the estimation of intracellular pH in resting and exercise human muscle samples.     

Surface-active and stimuli-responsive polymer--Si(100) hybrids from surface-initiated atom transfer radical polymerization for control of cell adhesion

A simple two-step method was developed for the covalent immobilization of atom-transfer radical polymerization (ATRP) initiators on the hydrogen-terminated Si(100) (Si-H) surface. Well-defined functional polymer-Si hybrids, consisting of covalently tethered brushes of poly(ethylene glycol) monomethacrylate (PEGMA) polymer, N-isopropylacrylamide (NIPAAm) polymer, and NIPAAm-PEGMA copolymers and block copolymers on Si-H surfaces, were prepared via surface-initiated ATRP. Kinetics study revealed that the chain growth from the silicon surface was consistent with a "controlled" process. Surface cultures of the cell line 3T3-Swiss albino on the hybrids were evaluated. The PEGMA graft-polymerized silicon [Si-g-P(PEGMA)] surface is very effective in preventing cell attachment and growth. At 37 degrees C [above the lower critical solution temperature (LCST, approximately 32 degrees C) of NIPAAm], the seeded cells adhered, spread, and proliferated on the NIPAAm graft polymerized silicon [Si-g-P(NIPAAm)] surface. Below the LCST, the cells detached from the Si-g-P(NIPAAm) surface spontaneously. Incorporation of PEGMA units into the NIPAAm chains of the Si-g-P(NIPAAm) surface via copolymerization resulted in more rapid cell detachment during the temperature transition. The "active" chain ends on the Si-g-P(PEGMA) and Si-g-P(NIPAAm) hybrids were also used as the macroinitiators for the synthesis of diblock copolymer brushes. Thus, not only are the hybrids potentially useful as stimuli-responsive adhesion modifiers for cells in silicon-based biomedical microdevices but also the active chain ends on the hybrid surfaces offer opportunities for further surface functionalization and molecular design.     

Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.     

Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature

Acrylic acid (AAc) has been utilized to introduce reactive carboxyl groups to a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). However, AAc introduction shifts the copolymer phase transition temperatures higher and dampens the steep homopolymer phase transition with increasing AAc content. We previously synthesized 2-carboxyisopropylacrylamide (CIPAAm) having both a similar side chain structure to IPAAm and a functional carboxylate group in order to overcome these shortcomings. In the present study, these copolymers, grafted onto cell culture plastic, were assessed for cell adhesion control using their phase transition. AAc introduction to PIPAAm-grafted surfaces resulted in excessive surface hydration and hindered cell spreading in culture at 37 degrees C. In contrast, CIPAAm-containing copolymer-grafted surfaces exhibited relatively weak hydrophobicity similar to both homopolymer PIPAAm-grafted surfaces as well as commercial ungrafted tissue culture polystyrene dish surfaces. Cells adhered and spread well on these surfaces at 37 degrees C in culture. As observed previously on PIPAAm-grafted surfaces, cells were spontaneously detached from the copolymer-grafted surfaces by reducing culture temperature. Cell detachment was accelerated on the CIPAAm copolymer-grafted surfaces compared to pure IPAAm surfaces, suggesting that hydrophilic carboxyl group microenvironment in the monomer and polymer is important to accelerate grafted surface hydration below the lower critical solution temperature, detaching cells.     

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells (Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1.Ca to E1'.Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu(37) is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37 degrees C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity ( approximately 0.75 micromol. nmol Ca-ATPase(-1).min(-1) at 15 microM Ca(2+)), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0 degrees C, using 1 microM MgATP, the K(Ca) values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37 degrees C. Measurements of the time course of phosphoenzyme formation at 0 degrees C, using 1 microM MgATP and 268 nM ionized [Ca(2+)], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca(2+) ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E.Ca to E'.Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca(2+) binding to the E1 intermediate.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.     

Hepatic drug metabolizing enzyme activity in the channel catfish, Ictalurus punctatus

1. Several pathways of drug metabolizing enzymic activity were measured in hepatic fractions of the channel catfish and rat using model substrates. The pathways examined included the O-demethylation of p-nitroanisole, microsomal ester hydrolysis of procaine and glucuronidation of p-nitrophenol, and the cytosolic acetylation of sulfamethazine and sulfation of 2-naphthol. Catfish liver preparations were incubated at both 25 degrees C and 37 degrees C. 2. The oxidative metabolism of p-nitrophenol was only one-eighth that of the rat at 37 degrees C and one-twelfth that of the rat at 25 degrees C. 3. Procaine ester hydrolysis was negligible in catfish microsomal preparations. 4. At 37 degrees C, p-nitrophenol glucuronidation was equivalent in catfish and rat microsomes. 5. Catfish cytosolic preparations exhibited N-acetyltransferase and arylsulfotransferase nearly comparable to those of the rat. 6. Rates of glucuronidation and sulfation were higher at 37 degrees C than at 25 degrees C in hepatic fractions of catfish.     

Degradation of chitosans with chitinase B from Serratia marcescens. Production of chito-oligosaccharides and insight into enzyme processivity

Family 18 chitinases such as chitinase B (ChiB) from Serratia marcescens catalyze glycoside hydrolysis via a mechanism involving the N-acetyl group of the sugar bound to the -1 subsite. We have studied the degradation of the soluble heteropolymer chitosan, to obtain further insight into catalysis in ChiB and to experimentally assess the proposed processive action of this enzyme. Degradation of chitosans with varying degrees of acetylation was monitored by following the size-distribution of oligomers, and oligomers were isolated and partly sequenced using (1)H-NMR spectroscopy. Degradation of a chitosan with 65% acetylated units showed that ChiB is an exo-enzyme which degrades the polymer chains from their nonreducing ends. The degradation showed biphasic kinetics: the faster phase is dominated by cleavage on the reducing side of two acetylated units (occupying subsites -2 and -1), while the slower kinetic phase reflects cleavage on the reducing side of a deacetylated and an acetylated unit (bound to subsites -2 and -1, respectively). The enzyme did not show preferences with respect to acetylation of the sugar bound in the +1 subsite. Thus, the preference for an acetylated unit is absolute in the -1 subsite, whereas substrate specificity is less stringent in the -2 and +1 subsites. Consequently, even chitosans with low degrees of acetylation could be degraded by ChiB, permitting the production of mixtures of oligosaccharides with different size distributions and chemical composition. Initially, the degradation of the 65% acetylated chitosan almost exclusively yielded oligomers with even-numbered chain lengths. This provides experimental evidence for a processive mode of action, moving the sugar chain two residues at a time. The results show that nonproductive binding events are not necessarily followed by substrate release but rather by consecutive relocations of the sugar chain.     

Continuous-flow determination of an alpha-amylase inactivator in fermentation samples using a ferricyanide reagent

A continuous-flow system has been developed in which pancreatic alpha-amylase is incubated with soluble starch at 37 degrees C. Reducing sugars being delivered at the "steady-state" hydrolysis of starch are dialyzed into a solution of alkaline ferricyanide. Ferricyanide is then reduced at 95 degrees C. The decrease in the absorbance of ferricyanide solution is recorded as "enzyme baseline." When samples containing alpha-amylase inactivator are introduced into the system, hydrolysis of starch is reduced according to the concentration of enzyme inactivator. The amount of inactivator in the samples is deduced from a standard curve of six standard concentrations using the Technicon Autoanalyzer II system manager in connection with a calculator for automated interpolation. This method allows to determine 40 samples/h with high sensitivity and precision (mean = 1.53 mg/liter; C.V. = 1.4%).