Candida antarctica lipase B-catalyzed synthesis of poly(butylene succinate): shorter chain building blocks also work

Lipase catalysis was successfully employed to synthesize high molecular weight poly(butylene succinate) (PBS). Attempts to copolymerize succinic acid with 1,4-butanediol were unsuccessful due to phase separation of the reactants. To circumvent this problem, monophasic reaction mixtures were prepared from diethyl succinate and 1,4-butanediol. The reactions were studied in bulk as well as in solution. Of the organic solvents evaluated, diphenyl ether was preferred, giving higher molecular weight products. After 24 h in diphenyl ether, polymerizations at 60, 70, 80, and 90 degrees C yielded PBS with M(n) of 2000, 4000, 8000, and 7000, respectively. Further increase in reaction time to 72 h resulted in little or no further increase in M(n). However, increasing the reaction time produced PBS with extraordinarily low M(w)/M(n) due to the diffusion and reaction between low-molecular weight oligomers and chains that occurs at a greater frequency than interchain transesterification. Time-course studies and visual observation of polymerizations at 80 degrees C revealed PBS precipitates at 5 to 10 h, limiting the growth of chains. To maintain a monophasic reaction mixture, the polymerization temperature was increased from 80 to 95 degrees C after 21 h. The result was an increase in the PBS molecular weight to M(w) = 38 000 (M(w)/M(n) = 1.39). This work paves the way for the synthesis of PBS macromers and polymers that contain variable quantities of monomers with chemically sensitive moieties (e.g., silicone, epoxy, vinyl). Furthermore, this study established the feasibility of using lipase catalysis to prepare polyesters from alpha,omega-linear aliphatic diethyl ester/diol monomers with less than six carbons.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Clostridium perfringens type A: in vitro system for sporulation and enterotoxin synthesis.

Polysomes were isolated from an enterotoxigenic strain of Clostridium perfringens during vegetative growth and at 1-h intervals after transfer into Duncan-Strong sporulation medium. During vegetative growth, about 67% of the ribosomes were in polysomal complexes. This proportion decreased to about 20% during the first 2 h in sporulation medium and then gradually increased to a maximum of 45% at 6 h. Ribosomes isolated from cells in vegetative or in sporulation phase could equally translate vegetative, sporulation, and natural viral R17 messenger ribonucleic acid with either vegetative or sporulation initiation factors. When polysomes were allowed to complete their nascent chains with labeled amino acids in vitro, most of the polypeptides synthesized by the vegetative phase and by the sporulation phase polysomes appeared to be identical. There were, however, notable differences upon further investigation. Specifically, when antiserum against the enterotoxin was reacted with the completed polypeptides, no counts were precipitated from the vegetative products. On the other hand, up to 12% of the total labeled protein was precipitated from the products obtained with the sporulation phase polysomes. Upon electrophoresis on sodium dodecyl sulfate, the putative enterotoxin synthesized in vitro ran as a major band with a molecular weight of 35,000, and as two minor bands with molecular weights of 17,000 and 52,000, respectively.     

Biogenesis of mitochondrial membranes in Neurospora crassa. Mitochondrial protein synthesis during conidial germination.

The conidia of Neurospora crassa entered logarithmic growth after a 1-h lag period at 30 degrees C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3-h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide-sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0-8-h-old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 mug cycloheximide/ml, increased from low levels in the cinidia to peak levels at 3-4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0-4-h-old cells, but declined after 15 min of incorporation in 6 and 8-h-old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12 200 to 80 000). Within the high-molecular-weight class, proteins of between 12 000 and 21 500 molecular weight were preferentially lavelled early in germination, whereas after 8 h of growth proteins of 27 500 to 80 000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000-molecular-weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.     

Protein translocation in the endoplasmic reticulum. Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins.

We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light. In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction. In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures. The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide. The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins. Both photolysis and alkaline carbonate extraction were required to produce the results. Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels. The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins. The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.     

Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts.

When isolated human fibroblast lysosomes are incubated with 4 microM [32P]phosphate at pH 7.0, orthophosphate is transported into lysosomes and is rapidly incorporated into low and high molecular weight products. We have characterized the high molecular weight (HMW) lysosomal material into which [32P]phosphate is incorporated and have found it to consist of long chains of inorganic polyphosphate based on the following observations. 1) greater than 97% of HMW 32P-lysosomal material is converted to [32P]orthophosphate when incubated with 1 N HCl for 20 min at 100 degrees C. 2) Incubation of HMW 32P-lysosomal material at pH 7.0 and 65 degrees C for 96 h results in the formation of [32P]trimetaphosphate, which is known to be produced only from linear chains of polyphosphate under these conditions. 3) HMW 32P-lysosomal material is resistant to degradation by proteinase K, ribonuclease, and deoxyribonuclease and extracts into the aqueous phase during phenol/chloroform extractions. 4) HMW 32P-lysosomal material displays heterogeneous mobility on polyacrylamide gels with most chains ranging in length from 100 to at least 600 phosphate residues. 5) HMW 32P-lysosomal material is partially hydrolyzed under alkaline conditions to yield a continuous ladder of polyphosphate species differing by one or several residues in length on polyacrylamide gels.     

Alpha-D-glucan-based dendritic nanoparticles prepared by in vitro enzymatic chain extension of glycogen

The recombinant amylosucrase from Neisseria polysaccharea was used to glucosylate glycogen particles in vitro in the presence of sucrose as the glucosyl donor. The morphology and structure of the resulting insoluble products were shown to strongly depend on the initial sucrose/glycogen weight ratio. For the lower ratio (1.14), all glucose molecules produced from sucrose were transferred onto glycogen, yielding a slight elongation of the external chains and their organization into small crystallites at the surface of the glycogen particles. With a high initial sucrose/glycogen ratio (342), the external glycogen chains were extended by amylosucrase, yielding dendritic nanoparticles with a diameter 4-5 times that of the initial particle. A partial crystallization of the elongated chains induced a "shrinkage" of the nanospheres. The synthesis of linear alpha-(1,4) chains occurred simultaneously, yielding semicrystalline fibrous entities. All products displayed a B-type crystal structure. The kinetics of chain elongation and aggregation were thoroughly investigated in order to explain how the action of amylosucrase resulted in such different structures. These results emphasize the potentiality of amylosucrase in the design of original carbohydrate-based dendritic nanoparticles.     

Subunit structure of human alpha 2-macroglobulin (alpha 2-MG) with respect to its interaction with trypsin.

SDS-polyacrylamide gel electrophoresis of a recently prepared alpha 2-macroglobulin solution showed only the polypeptide chains of 190,000 molecular weight. Reduction-alkylation of this preparation followed by gel-filtration on a Sephadex G-200 column in 5.2 M guanidine hydrochloride was unable to separate a fraction of 83,000 molecular weight as previously described. Nevertheless, after incubation of a mixture alpha 2-macroglobulin-trypsin during 45 minutes at 37 degrees C, approximately 60 per cent of the preparation were converted in a component with 83,000 molecular weight as detected in SDS polyacrylamide gel. That component was isolated on Sephadex G-200 in guanidine hydrochloride and corresponds to the subunit, fraction II. According to the results of the present work together with those of previous studies, it can be assumed that alpha 2-MG is a 780,000 molecular weight protein (19S) formed of two half-molecules of equal weight (11-12S). The half-molecule contains two polypeptide chains of 180,000-190,000 molecular weight, each of them having, in its middle, a specific region particularly susceptible to attack by proteases.     

Acetylation of wheat straw hemicellulose B in a new non-aqueous swelling system

The acetylation of wheat straw hemicellulose B was carried out in a homogeneous N,N-dimethylformamide and lithium chloride system with acetic anhydride using 4-dimethylaminopyridine as a catalyst. The degree of substitution of hemicellulose B acetates ranged between 0.59 and 1.25 as a function of experimental conditions. Under an optimum condition (85°C, 60 h), approximately 75% of the free hydroxyl groups in native hemicellulose B were acetylated. The molecular weight measurements (31,890–34,090 g mol⁻¹) showed a controllable degradation of hemicellulose B chains during the reactions at temperature 60–85°C and duration of 2–60 h. It was found that the thermal stability of the products was increased by chemical modification.     

超声波处理对柴胡多糖提取率、微观形貌特征及生物活性的影响

为了研究超声波辅助提取柴胡多糖的过程中超声波对柴胡多糖的提取率及外观形貌、生物活性的影响,在单因素（超声波功率、超声波作用时间、料液比）的基础上,通过正交实验确定超声波辅助提取最佳工艺条件,并与传统提取方法所得到的结果进行了比较：利用原子力显微镜（AFM）研究超声波作用对柴胡多糖的形貌特征的影响;用邻二氮菲-金属铁离子-H2O2体系检测柴胡多糖对羟基自由基的清除作用。实验结果表明：超声波辅助提取的最佳提取条件为超声功率360W,超声时间15min,料液比1：35,水浴温度90℃,水浴时间1h,提取率为2．58％．所获得的柴胡多糖（SBR）的纯度为44．14％,与传统提取方法相比．不仅节约了时间,而且提高了提取效率。原子力显微镜观察的结果表明,柴胡多糖分子主要以螺旋结构形态存在,超声波作用使得柴胡多糖的分子降解成较小的分子片段。柴胡多糖能有效清除羟自由基,在相同浓度下SBP。的清除效果要优于水提柴胡粗多糖（WBP0）,且质量浓度在80～100ug／mL的范围清除效果最佳,并高于同浓度下抗坏血酸的清除效果。     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Preparation and hydrolytic degradation of semi-interpenetrating networks of poly(3-hydroxyundecenoate) and poly(lactide-co-glycolide)

Semi-interpenetrating networks (semi-IPNs), where poly(lactide-co-glycolide) (PLGA) molecules were entrapped in the crosslinked matrices of poly(3-hydroxyundecenoate) (PHU), were prepared by irradiating homogeneous solutions of PHU and PLGA in chloroform with UV light. Attenuated total reflectance infrared spectroscopy showed that the PLGA chains were entrapped in PHU networks. The semi-IPNs showed enhanced mechanical strength as the PLGA content increased. The semi-IPNs were incubated at 37 °C in a 0.01N NaOH solution, and the extent of hydrolytic degradation was investigated by monitoring changes in various parameters such as water uptake, pH, mass, and morphology. Hydrolysis of semi-IPNs were significantly affected by the presence of PLGA. A semi-IPN prepared from a 9:1 (by weight) mixture of PHU and PLGA lost 25% of its original weight in 12 weeks while a PHU sample containing no PLGA lost only 5% of its weight during the same period under identical conditions. The hydrolysis was most likely accelerated when the pH of the medium was lowered by the hydrolyzed products of PLGA, 2-hydroxyalkanoic acids. These results showed that hydrolysis of PHA could be enhanced by incorporating a second component that lowered the pH of the hydrolysis system.     

Molecular changes associated with proteolysis of bovine factor V by thrombin.

Native factor V contains two major polypeptide chains, h and 1. The molecular weights determined by gel electrophoresis in the presence of sodium dodecylsulfate and dithiothreitol (125 000 and 73 000) are in reasonable agreement with those obtained by gel filtration in 5 M guanidine-HC1 (125000 and 64000). Exposure of factor V to thrombin results in cleavage of the heavier chain to an altered form with a molecular weight of 87000. The other fragment of this proteolytic reaction appears to be a carbohydrate-rich piece, which migrates abnormally slowly on gel electrophoresis conducted under denaturing and reducing conditions. Both proteolytic cleavage products remain associated with the light chain during the purification of factor V. The 87000-Mr fragment is present in samples of factor V which are isolated by immunoprecipitation of blood obtained from a single animal by venous catheter. This finding suggests that some proteolysis may occur in vivo. In contrast, the molecular weight of the light chain is unaltered after thrombin proteolysis of either purified factor V or thrombin-treated plasma.     

Glycosaminoglycan free chains. External plasma membrane components distinct from the membrane proteoglycans

Heparan sulfate and chondroitin sulfate glycosaminoglycans of BALB/c 3T3 fibroblasts, metabolically labeled with [3H]glucosamine and [35S]sulfate precursors, are resolved by preparative Sepharose CL-4B chromatography into distinct products, the proteoglycans and the glycosaminoglycan free chains, the latter resistant to appreciable molecular weight shift upon alkaline borohydride reduction. The in situ localization of these cell layer molecules was probed with glycosaminoglycan degrading enzymes (lyases) of bacterial origin, which were used to digest isotopically prelabeled monolayer cultures prior to extraction with a nonionic detergent in the presence of protease inhibitors. Most of the total cellular complement of glycosaminoglycan free chains, in addition to the proteoglycans, proved accessible to the lyases under conditions which did not appreciably affect cell viability or morphology. Because these results were also obtained under low temperature (4 degrees C) conditions and in the presence of phenylarsine oxide, a sulfhydryl reagent that irreversibly inhibits endocytosis, the effects of the lyases are not dependent upon internalization by the cells. The cellular production and cell surface expression of the glycosaminoglycan free chains were not materially altered when lysosomal function was pharmacologically inhibited, confirming that the free chains are not intracellular intermediates in the lysosomal degradation pathways of proteoglycans. Contrary to the prevailing model, our observations establish that, at least in the cell line under study, glycosaminoglycan free chains are located on the external leaflet of the plasma membrane, as such suggesting that these products are biologically active components of cell surfaces.     

Synthesis and study of cross-linked chitosan-N-poly(ethylene glycol) nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan. Poly(ethylene glycol) dicarboxylic acid was used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. The prepared nanosystems were stable in aqueous media. The structure of the products was determined by nuclear magnetic resonance (NMR) spectroscopy, and the particle size was identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that biodegradable cross-linked chitosan nanoparticles experienced considerable swelling because of the length and flexibility of the cross-linking agent. The aqueous solutions or dispersions of nanoparticles were stable and clear or mildly opalescent systems depending on the ratio of cross-linking and molecular weight of chitosan, findings consistent with values of transmittance above 75%. Particle size measured by TEM varied in the range of 4-24 nm. In the swollen state, the average size of the individual particles measured by DLS was in the range of 50-120 nm depending on the molecular weight of chitosan and the ratio of cross-linking.     

Cell-free translation products of basement membrane RNA from the EHS tumor

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of alpha 1(IV) and alpha 2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170,000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature alpha 1(IV) and alpha 2(IV) chains in the EHS tumor matrix.     

Synthesis and characterization of new permanently charged poly(amidoammonium) salts and evaluation of their DNA complexes for gene transport

A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected.     

Influence of in vitro hydrolytic degradation on the morphology and crystallization behavior of poly(p-dioxanone)

We have studied the hydrolytic degradation of high molecular weight poly(p-dioxanone), PPDX, sutures. The samples were degraded either in distilled water or in a phosphate buffer at 37 degrees C, and the starting viscosity-average molecular weight was 130 kg/mol. The hydrolytic degradation of PPDX occurs in an approximate two stage process where the amorphous regions of the sample are attacked faster than the crystalline regions of the sample. The changes experienced by the samples as degradation proceeded were successfully monitored by viscosimetry, differential scanning calorimetry (DSC), weight loss, pH changes, and scanning electron microscopy (SEM). Polarized optical microscopy (POM) observations performed on PPDX films revealed that PPDX crystallizes in spherulites whose detailed morphology depends on the supercooling employed during isothermal crystallization. Changes in the spherulitic morphology as molecular weight is reduced are only pronounced when the molecular weight is equal or lower than 8 kg/mol. The dependence of lamellar thickness as a function of isothermal crystallization temperature was examined by atomic force microscopy (AFM) in thin films of PPDX together with melting point data obtained by DSC. Through the use of the Thomson-Gibbs equation, we obtained a value of 166 erg/cm2 for the fold surface free energy of PPDX. This value is in the same range as those obtained previously for similar linear polyesters. The lamellar thickness, as well as the melting point, was found to have a small decreasing dependence with the molecular weight of the samples.     

Purification and properties of alpha-ketoglutarate reductase from Micrococcus aerogenes

Micrococcus aerogenes grown in media containing glutamate has high levels of glutamate dehydrogenase and alpha-ketoglutarate reductase. The latter enzyme catalyzes the reversible reduction of alpha-ketoglutarate to alpha-hydroxyglutarate in the presence of reduced nicotinamide adenine dinucleotide (NADH). The enzyme has a high specificity for both substrates in either direction and displays Michaelis-Menten kinetics at moderate substrate concentrations. K(m) values of 0.12 to 0.17 mm alpha-ketoglutarate and 0.3 mm NADH for the forward reaction were calculated from data obtained at low substrate concentrations. At high concentrations, this reaction was inhibited by both substrates. The reverse reaction, which proceeded at 0.1 to 0.2 times the rate of the forward reactions, was inhibited by one of the products, alpha-ketoglutarate. K(m) values for the substrates of this reaction were 10 mm for alpha-hydroxyglutarate and 1 mm for nicotinamide adenine dinucleotide. alpha-Ketoglutarate reductase has a molecular weight of 7.5 x 10(4) to 8.2 x 10(4) and is composed of identical polypeptide chains with a molecular weight of 3.6 x 10(4) to 3.8 x 10(4).