Low Molecular Weight Substrates Research Articles

Optimization of the production of triabin, a novel thrombin inhibitor, in High Five™ insect cells infected with a recombinant baculovirus.

The isolation of a new type of thrombin inhibitor, called triabin, from the saliva of the hematophagous bug Triatoma pallidipennis, has recently been described. In the in vitro platelet aggregation inhibition assay triabin has a similar potency as the thrombin inhibitor hirudin now in phase III clinical trials. However, in another in vitro assay using a low molecular weight substrate for thrombin, triabin does not inhibit thrombin completely even at 6 fold higher molar doses in comparison with hirudin. This means that triabin has a novel mode of action towards thrombin making triabin into an interesting candidate as a therapeutic agent. Recently it has been shown that a recombinant baculovirus can be efficiently used for the triabin production in insect cells and that the yields in adherent cultures of High Five™ cells (approx. 20 mg l-1) were about 7 fold higher than in adherent cultures of Sf9 cells (approx. 3 mg l- 1). To optimize the triabin yield from the baculovirus/insect cell expression system, experiments were performed with suspension adapted cultures of High Five™ cells to investigate the effects of the state of the host cell, of the multiplicity of infection, of the cell density at the time of infection and of supplementation of the medium with nutrients and oxygen. Triabin yields of up to 200 mg l-1, as determined by an activity assay, could finally be obtained here.

Salicylate Stimulates the Degradation of High-Molecular Weight Polycyclic Aromatic Hydrocarbons by Pseudomonas saccharophila P15

Pseudomonas saccharophila P15 was isolated from soil contaminated with polycyclic aromatic hydrocarbons (PAH) and previously was reported to degrade a variety of low- and high-molecular weight PAH. Strain P15 grows on phenanthrene by a known pathway in which salicylate is an intermediate. Preincubation with phenanthrene and downstream intermediates through salicylate stimulated PAH dioxygenase activity and initial rates of phenanthrene removal, suggesting that salicylate is the inducer of these activities. Salicylate also greatly enhanced initial rates of removal of fluoranthene, pyrene, benz[a]anthracene, chrysene, and benzo[a]pyrene, high-molecular weight substrates that strain P15 does not use for growth. The specific rate of removal of benzo[a]pyrene was at least 2 orders of magnitude lower than that of the four-ring compounds and nearly 5 orders of magnitude lower than that of phenanthrene. The mineralization of phenanthrene, benz[a]anthracene, chrysene, and benzo[a]pyrene was stimulated by preincubation with phenanthrene or salicylate, although significant mineralization of phenanthrene, benz[a]anthracene, and chrysene occurred in uninduced cultures. Further experiments with chrysene indicated that chrysene does not appear to induce its own mineralization. Our results suggest that P. saccharophila P15 expresses a low level of constitutive PAH metabolism which is inducible to much higher levels and that high-molecular weight PAH metabolism by this organism is induced by the low-molecular weight substrate phenanthrene and by salicylate.

Functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] for protein modification.

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different functional end groups, namely carboxyl, methyl ester, hydrazide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2'-Azobisisobutyronitrile (AIBN) was used as an initiator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mercaptopropionic hydrazide, and 2-mercaptoethylamine were used as chain-transfer agents. The semitelechelic polymers have been characterized by end-group analysis, size-exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans and the initiator on the molecular weight of the polymers have been investigated. The higher the concentration of mercaptan, the lower the molecular weight of ST-PHPMA. The concentration of initiator did not have a significant effect on the molecular weight of the semitelechelic polymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, alpha-chymotrypsin, has been modified with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu and the conjugates characterized by MALDI-TOF MS. The activity of modified chymotrypsins toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was slightly lower than the activity of the native enzyme. The cleavage of a low molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotrypsins was dependent on their structure. Whereas the activity of the amino group modified chymotrypsins was higher than that of the native enzyme, the activity of carboxyl-modified chymotrypsins was lower than that of the native enzyme. In summary, the data seem to indicate that ST-PHPMA is an effective protein-modifying agent.

Activity and inhibition of elastase in GCF.

The aim of this study was to investigate the presence of free elastase in GCF Samples were taken from inflamed sites in 12 subjects with gingivitis alone and from inflamed sites with and without tissue destruction in 19 patients having periodontitis. Elastase activity was measured with a low molecular weight substrate. To distinguish between free elastase and elastase bound to alpha-2-macroglobulin (A2MG), an excess of alpha-1-antitrypsin (A1AT) was added to the samples. The activity that could be inhibited by A1AT was considered as free elastase, and the uninhibited activity as derived from the elastase-A2MG complex. The elastase-A1AT complex was measured with an ELISA. Free elastase was found in almost all samples, but both the total amount and the proportion of free elastase were higher in samples from sites showing destruction. The elastase-A2MG complex was also increased in sites with tissue destruction, while there was no significant difference in the amount of A1AT complex between the 3 categories of sites. In conclusion, our study clearly reveals free elastase in GCF The elevated levels of free elastase in sites showing tissue destruction seem to be due to a combination of increased release of elastase and an inactivation of A1AT.

Conjugation of Trypsin by Temperature-Sensitive Polymers Containing a Carbohydrate Moiety: Thermal Modulation of Enzyme Activity

Novel temperature-sensitive polymers containing glucose units in their backbone were synthesized and covalently conjugated to trypsin. A series of copolymers based on N-isopropylacrylamide (NIPAAm) and glucosyoxylethyl methacrylate (GEMA) were prepared by using 4,4'-azobis(4-cyanovaleric acid) as an initiator, which resulted in one terminal carboxylic acid group per polymer chain. The polymers were conjugated to primary amine groups of trypsin with water-soluble carbodiimide as a coupling agent, which led to a star-shaped conformation. The polymer-enzyme conjugation was confirmed and characterized by size exclusion and reversed-phase chromatography. Almost of all amine groups in trypsin available for the conjugation were consumed and, consequently, a very dense layer of copolymers was actually coated around the enzyme surface. The conjugated enzymes exhibited reversible precipitation/resolubilization behaviors over a wide range of temperatures, depending on the content of GEMA in the copolymer. They also demonstrated no detectable self-digestion (autolysis) process, but the unconjugated enzyme showed very severe autolysis that led to a rapid inactivation in aqueous solution. When bovine serum albumin was used as a substrate, the protein substrate was not attacked by the conjugated enzyme, but completely digested by the unconjugated enzyme. This result was presumably caused by a steric repulsion process of the attached polymer chains around the enzyme toward the protein substrate. However, the enzyme retained sufficient activity against a low molecular weight substrate. Interestingly, the conjugated enzymes demonstrated very peculiar enzyme activity-temperature profiles, with two apparent optimal temperatures, indicating that a temperature-controlled collapse and flocculation of the copolymers around the enzyme surface modulated the mass transfer rates of substrate to the active site of the enzyme. The conjugated enzymes also exhibited improved thermal stability with increasing the amount of carbohydrate units in the polymer chain.

Cathepsin B, α2‐macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients

Gingival crevicular fluid (GCF) was collected from 16 molar and premolar sites in each of 20 chronic periodontitis patients before and after periodontal therapy using filter paper strips. These were eluted individually into buffer for determination of cathepsin B and its endogenous inhibitors, alpha2-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, alpha2-macroglobulin by enzyme-linked immunosorbent assay and cystatin activity by inhibition of papain. Total amounts of enzyme and inhibitor per GCF sample decreased after treatment and correlated positively with pocket depth and gingival, bleeding and plaque indices. These comparisons were nearly always statistically significant for pooled site data and sometimes so for mean patient values. The amounts of alpha2-macroglobulin and cystatin were greater than those of cathepsin B and, surprisingly, enzyme and inhibitor levels correlated positively with each other. Experiments with purified reagents, however, demonstrated that the cathepsin B: alpha2-macroglobulin complex was still active against the low molecular weight substrate and that cystatin levels in GCF are probably insufficient to inhibit the enzyme substantially These factors may explain why GCF cathepsin B activity reflects the clinical status of periodontal lesions and has been identified in another study as a promising indicator of disease progression.

Contribution of Pro212-Ile276 Sequence of Human Protein C to its Anticoagulant and Profibrinolytic Activity

Activated protein C exhibits strong anticoagulant and profibrinolytic properties. The Pro212-Ile276 fragment of a heavy chain of the protein molecule is located in the nearest neighborhood of the catalytic domain. It was found that polyclonal antibodies against this fragment recognize the sequence in the native molecule. To determine the contribution of the fragment in the anticoagulant and profibrinolytic activities of the enzyme, competitive inhibition analyses were performed. We found that amidolytic activity of the enzyme was inhibited by the recombinant Pro212-Ile276 fragment in a dose-dependent manner. Also, the fusion protein prolonged the time of clot lysis in a micro-clot lysis assay. The presence of the recombinant protein fragment did not influence the reaction of activated protein C with factor Va, neither the reaction of the enzyme with its specific inhibitor. We conclude, that Pro212-Ile276 region of protein C can not be identified with the binding pocket of the enzyme, but might be a binding site for small, low molecular weight substrates like chromogenic substrate.

Biosynthesis of (2)H-Labeled Phenylalanine by a New Methylotrophic Mutant Brevibacterium methylicum.

The biosynthesis of (2)H-labeled phenylalanine was done by converse of low molecular weight substrates ([U-(2)H]methanol and (2)H2O) in a new RuMP facultative methylotrophic mutant Brevibacterium methylicum. To make the process work, adapted cells with improved growth characteristics were used on minimal medium M9 with the maximum content of (2)H-labeled substrates. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in addition to the main product of biosynthesis. Electron impact mass spectrometry of methyl esters of the N-Dns-amino acid mixture obtained after the chemical derivatization of growth medium with dansyl chloride and diazomethane, was done to calculate the deuterium enrichment of the amino acids synthesized. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium enrichment were detected in all presented molecules-for phenylalanine the enrichment was six, leucine/isoleucine-five, valine-five, and alanine-three deuterium atoms.

Structures and functions of ribonucleases

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.

The direct or MIT route for nitrogen immobilization: A 15N mirror image study with leucine and glycine

Current models of nitrogen mineralization implicitly assume that all the N in organic substrates is mineralized to NH 4 + + before assimilation by growing microorganisms — the so-called MIT hypothesis. In theory, however, N mineralization during the decomposition of low molecular weight substrates such as amino acids can occur by a direct route in which the substrate is assimilated into the cell, deaminated, and only the surplus released into the soil NH 4 + pool. The decomposition in soil of the amino acids leucine and glycine was studied, using the techniques of 15N isotope dilution and enrichment, to determine whether the direct route of N assimilation was occurring in soil. The results showed clearly that the conventional MIT route was not operative and were consistent with the direct route. The relative proportions of N from leucine and glycine mineralized into the NH 4 + pool were in close agreement with predictions based on the direct hypothesis. Results from other published work on amino acid decomposition, reinterpreted using these techniques, were also consistent with the direct route.

Arylsulfatase activity in soil and soil extracts using natural and artificial substrates

The arylsulfatase activity of soil and humic arylsulfatase complexes extracted from soil were measured using the substrates p-nitrophenyl sulfate and low molecular weight (500–10000) soil ester sulfate compounds. Soil samples from the Aphorizon of a Podzol from S-amended wheat plots and a Regosol from dykeland hayfield plots were investigated. Soil arylsulfatase activity (assayed with p-nitrophenyl sulfate) in the fall was significantly higher than spring samples; however, no seasonal differences were observed when humic-arylsulfatase complexes were assayed with p-nitrophenyl sulfate. The discrepancy between arylsulfatase activity in soil and soil extracts was probably due to inhibitors which were found in soil materials. These results appear to support the theory that abiotic arylsulfatase is a relatively stable and persistent component of soil. There was a marked difference in the response by humic-arylsulfatase complexes to the artificial substrate p-nitrophenyl sulfate and natural low molecular weight soil substrates. Humic-arylsulfatase complexes hydrolysed 35–80% of added low molecular weight substrates depending on the treatment. The molecular size, concentration, and chemical composition of the low molecular weight ester sulfate compounds affected hydrolysis of the low molecular weight substrates. The response by humic-arylsulfatase complexes to the chromogenic ester sulfate, p-nitrophenyl sulfate did not reflect the ability of these complexes to hydrolyse natural soil substrates. In an experiments we examined arylsulfatase activity and soil S status in relation to the total S in plant tissue and grain from wheat plants grown in the Podzol. Tissue S was more strongly associated with soil S than the wheat grain. Hydriodic acid-S, Ca(H2PO4)2-extractable sulfate, and hydrolysable ester sulfates in the high molecular weight (>10000) and low molecular weight (500–10000) fractions of soil organic matter extracts were strongly positively correlated with tissue S. Arylsulfatase activity in soil and humic-arylsulfatase extracts assayed with p-nitrophenyl sulfate were also strongly correlated with tissue S, while humic-arylsulfatase activity assayed with the low molecular weight substrate was negatively correlated with tissue S.

Characterization and purification of fucosyltransferases from the cytosol of rat colon

The occurrence and baseline characteristics of fucosyltransfernses (α-1,2, α-1,3 and α-1,4) in the cytosol (soluble) and pellet - (membrane-bound) of rat colon have been studied since the fucosylation process is known to alter in colon pathology. All enzymes studied in the colon pellet had higher activity when compared to the cytosol. The colon pellet α-1,3 fucosyltransferase preferred desialylated α 1-acid glycoprotein as acceptor substrate. Both soluble and membranebound enzymes, α-1,2 and α-1,3 fucosyltransferases, required Mn 2+, Mg 2+ and Ca 2+ for maximum activity but were inactivated by Cu 2+ ions. Both soluble α-1,2 and α-1,3 fucosyltransferases showed optimal activity at pH 6.0, whereas the optimum for their membrane-bound activities were at pH 5.8 and 6.2, respectively. Furthermore, a soluble α-1,3 fucosyltransferase from rat colon was purified and during purification the co-presence of α-1, 3 4 fucosyhransferase was detected. The acceptor of preference for the purified soluble α-1,3 fucosyltransferase was desialylated glycoprotein while low molecular weight substrates were poor acceptors. Both the purified fucosyltransferases were inhibited by N-ethylmaleimide. The M r values determined by SDS-PAGE electrophoresis of α-1, 3 4 fucosyltransferase and of α-1,3 fucosyltrangferase were 68,780 and 40,680, respectively. In conclusion, based on their properties, the purified soluble colon α-1,3 fucosyttransferase appeared to be of plasma-type (or FT-I) while the soluble α-1,3/4 fucosyltraroferase corresponded to Lewis-type or FT-III.

Lysozyme substrates.

The natural substrate of lysozyme is the rigid layer of bacterial cell walls, the murein (peptidoglycan), which is a gigantic polymer of (GlcNAc-MurNAc)n polysaccharide strands crosslinked through short peptide bridges at the lactyl groups of the muramic acid residues. Thus, lysozyme lyses bacteria by degrading their protective exoskeleton, the murein sacculus. The high molecular weight murein is thereby hydrolysed to low molecular weight muropeptides, a process that can be followed quantitatively by different methods. However, due to the insolubility of the murein sacculus, the enzyme kinetics are rather complex. Therefore, a variety of different low molecular weight substrates have been prepared, both murein degradation products and synthetic compounds. These substrates allow a better characterization of the binding and catalytic mechanism of lysozyme. In addition, they are used in various photometric, isotopic and immunological lysozyme assays.

歯周病り患部位におけるｓｅｃｒｅｔｏｒｙ ｌｅｕｋｏｃｙｔｅ ｐｒｏｔｃａｓｅ ｉｎｈｉｂｉｔｏｒ（ＳＬＰＩ）の動態

The present investigation was designed to clarify the possible relationship between the secretory leukocyte protease inhibitor (SLPI) in gingival crevicular fluid (GCF) and the periodontal clinical parameters or the presence of periodontopathic bacteria in subgingival plaque.SLPI levels in GCF were determined with a commercially available ELISA system and the neutrophil elastase (NE) activity in GCF was measured with a low molecular weight substrate S-2484. Subgingival plaque samples were tested for the presence of eight periodontopathic bacteria by DNA probe analysis.In thirty two examined sites from ten patients with periodontal maintenance care, the SLPI levels were positively associated with the GCF volume and the presence of gingival inflammation. Statistically significant negative correlations were found between SLPI levels and the presence of Porphyromonas gingivalis and Treponema denticola, whereas, the NE activity increased in the presence of the bacteria.The results demonstrated that the bacteria with the capacity to produce proteolytic enzymes degraded or inactivated the protease inhibitor, suggesting that the proteolytic damage of periodontal tissue might be caused by the decrease of active protease inhibitor.

Methanogenesis in the sediments of an Australian freshwater wetland: Comparison with aerobic decay, and factors controlling methanogenesis

Sediment oxygen demands and water-atmosphere methane emissions of a highly productive, freshwater wetland on the floodplain of the River Murray in south-eastern Australia were quantified over a 14 month period in 1993–1994. Total sediment oxygen demands ranged from 1.3 to 3.3 mmol m −2 h −1, of which < 3 to 90% was due to chemical oxygen demand. Methane emissions ranged from < 0.01 mmol m −2 h −1 in winter to 2.75 mmol m −2 h −1 in summer. Methanogenesis accounted for at least 60% of the combined aerobic and methanogenic carbon flux in sediments from Eleocharis sphacelata beds, and at least 30% and 40% of the combined flux in sediments from Myriophyllum sp. beds and Vallisneria gigantea beds, respectively. In vitro incubations, using additions of sulfate and of molybdate, failed to indicate unequivocally competition for substrates between sulfate-reducing and methanogenic bacteria. However, in vitro methanogenesis was strongly inhibited by nitrate, suggesting an interaction between benthic methanogens and denitrifying or other nitrate-reducing bacteria. Fe 3+ decreased in vitro methanogenesis by 16–49% during January, February and March 1994; oxidation of organic matter at the expense of the reduction of ferric ions could be a significant route for detritus processing in Eleocharis-bed sediments in the warmer months. Methanogenesis was increased consistently by additions of some low molecular weight substrates, such as acetate, but not by others, such as methanol, propionate and trimethylamine. Complex polymeric substrates, such as cellulose, starch and aquatic plant matter, increased in vitro methanogenesis rapidly and markedly. Despite this, no relationship between methane emissions and benthic cellulase activity was observed in the field. Methanogenesis was strongly temperature dependent, being maximal at 30 to 40°C and minimal at 5°C, thereby explaining the strong seasonality observed in methane emissions in situ.

Methanogenesis in the sediments of an Australian freshwater wetland: Comparison with aerobic decay, and factors controlling methanogenesis

Sediment oxygen demands and water-atmosphere methane emissions of a highly productive, freshwater wetland on the floodplain of the River Murray in south-eastern Australia were quantified over a 14 month period in 1993–1994. Total sediment oxygen demands ranged from 1.3 to 3.3 mmol m−2 h−1, of which < 3 to 90% was due to chemical oxygen demand. Methane emissions ranged from < 0.01 mmol m−2 h−1 in winter to 2.75 mmol m−2 h−1 in summer. Methanogenesis accounted for at least 60% of the combined aerobic and methanogenic carbon flux in sediments from Eleocharis sphacelata beds, and at least 30% and 40% of the combined flux in sediments from Myriophyllum sp. beds and Vallisneria gigantea beds, respectively. In vitro incubations, using additions of sulfate and of molybdate, failed to indicate unequivocally competition for substrates between sulfate-reducing and methanogenic bacteria. However, in vitro methanogenesis was strongly inhibited by nitrate, suggesting an interaction between benthic methanogens and denitrifying or other nitrate-reducing bacteria. Fe3+ decreased in vitro methanogenesis by 16–49% during January, February and March 1994; oxidation of organic matter at the expense of the reduction of ferric ions could be a significant route for detritus processing in Eleocharis-bed sediments in the warmer months. Methanogenesis was increased consistently by additions of some low molecular weight substrates, such as acetate, but not by others, such as methanol, propionate and trimethylamine. Complex polymeric substrates, such as cellulose, starch and aquatic plant matter, increased in vitro methanogenesis rapidly and markedly. Despite this, no relationship between methane emissions and benthic cellulase activity was observed in the field. Methanogenesis was strongly temperature dependent, being maximal at 30 to 40°C and minimal at 5°C, thereby explaining the strong seasonality observed in methane emissions in situ.

Synthesis of Porous Polymer Carrier and Immobilization of α-Chymotrypsin

A porous resin, acrolein–vinyl acetate–divinyl benzene copolymer, was synthesized by radical suspension polymerization of acrolein with vinyl acetate and divinylbenzene in the presence of a pore-creating agent, toluene. A porous polymer carrier containing both anilino and hydroxy groups was obtained by further reaction of the resin with p-nitroaniline, then reduction of the nitro group to amino group. α-Chymotrypsin was immobilized on the porous polymer carrier by the glutaraldehyde–crosslinking method or diazo-coupling method. The activity recovery and relative activity of the immobilized α-chymotrypsin were determined when l-tyrosine ethyl ester and casein were selected as low and high molecular weight substrates, respectively. At the same time, the stability, resistance against inhibition, the Michaelis constant Km, and reusability of the immobilized enzyme were also investigated. The results indicated that the immobilized α-chymotrypsin had not only higher relative activity, but also remarkable stability and better reusability. In addition, the immobilized α-chymotrypsin had stronger resistance against Cu2+ inhibition.

Protein kinase C substrates in corneal epithelium during wound healing: the phosphorylation of growth associated protein-43 (GAP-43)

Protein kinase C (PKC) plays an important role in regulating cell growth. In the cornea, α-PKC activity increases during wound-healing. This activition of PKC will result in the increased phosphorylation of specific PKC substrates. In this study, several PKC substrates of relative low molecular weight were identified and characterized in cytosol and membrane preparations obtained from rabbit corneal epithelium before and during wound-healing. Corneal epithelium proteins were phosphorylated by endogenous PKC and by α-PKC isolated from rabbit brain, and then analysed using SDS-PAGE. In cytosol, PKC substrates with apparent molecular weights of 20, 25, 30, 35, 50 and 55 kDa were phosphorylated by PKC. The phosphorylation of the substrates increased 3 and 7 days after total deepithelialization, due to the increase in α-PKC activity after wounding. However, when brain α-PKC was used as the exogenous source of PKC, there was a protein concentration-related decrease in the phosphorylation of corneal epithelium substrate after injury. This decreased phosphorylation of PKC substrates was inhibited by okadaic acid, a specific phosphatase inhibitor. The results suggest that an activated protein phosphatase takes part in controling the phosphorylation of PKC substrates during wound-healing. In the membrane fraction, a 60 kDa protein was phosphorylated by α-, β- and γ-PKC isoenzymes and was identified by Western blot as growth associated protein-43 (GAP-43), a protein kinase C substrate involved in axon regeneration. GAP-43 concentration increased 3 and 7 days after wounding as did its phosphorylation by α-PKC. These findings suggest a role for the protein in the innervation process in corneal epithelium after injury.

Measurement of the Hemoglobin Concentration in Deoxyhemoglobin S Polymers and Characterization of the Polymer Water Compartment

Biological polymers contain freely exchangeable water within intermolecular crevices with restricted access to large extrapolymer solutes. Our recent studies highlighted large osmotic effects of such polymer water compartments (PWCs), and their substantial physiological and pathophysiological relevance. The size and accessibility of the PWC are critical parameters determining the polymers' osmotic properties. We report here a new experimental approach to investigate these parameters in deoxyhemoglobin S polymers. The size of the PWC is inversely related to the deoxyhemoglobin S concentration in the polymer ( C P). Only an approximation of C P(≈69 g/dl) was previously available. By analyzing the distribution s of soluble hemoglobin and a large molecular weight (MW) marker ( 14C-dextran, MW ≈ 70kDa) in the supernatant and pellet of centrifuged gels, we obtained a reproducible value of C P1 54.7(±0.7)g/dl. This indicates that 60% of the polymer is composed of a water compartment inaccessible to soluble Hb and other non-interactive macromolecules. The accessibility properties of this PWC to smaller molecules were explored with markers of different MW. Non-interactive markers with MW < 200 kDa diffused freely in the PWC, whereas those with 300 kDa < Mw < 1000 KDa showed partial exclusion. Higher MW markers were generally excluded, except molecules with elongated (rather than spherical) shapes or possible interactivity with hemoglobin. These result predict that dense sickle cells would significantly dehydrate on deoxygenation, generating a PWC of up to 60% to 80% of the cell water. Soluble enzymes would concentrate in the residual cytosol. For osmotic equilibrium, most of the ions and low MW substrates would concentrate in the PWC. Oxygenation-deoxygenation would thus cause dynamic oscillations in cell hydration and between states of single and double cytoplasmic water phases, the water phases, the letter with a substantially altered internal environment. The relevance of such oscillations to the membrane and metabolic abnormalities of dense sickle cells require further investigation.

Isolation and partial characterization of an intracellular esterase from Enterococcus faecium ACA-DC 237

An intracellular esterase from Enterococcus faecium ACA-DC 237, isolated from Greek Feta cheese, was purified on DEAE-cellulose and Sephadex G-150. The enzyme had a molecular weight of 45 000, with an optimum activity on 4-nitrophenyl butyrate at pH 8.0 and at 35°C, with K m = 0.85 mM. The esterase was capable of degrading synthetic substrates of low and medium molecular weight (C2 to C12). It was inactivated by PMSF; sulfhydryl group reagents and metal chelators had little or no effect on enzyme activity.