Intact Enzymes Research Articles

Nitric oxide sensitive guanylyl cyclase activity decreases during cerebral postnatal development because of a reduction in heterodimerization

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co-precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.

Nitrite directly vasodilates hypoxic vasculature via nitric oxide‐dependent and ‐independent pathways

It is postulated that nitrite requires reduction to nitric oxide in order to exert its relaxant effect upon isolated hypoxic vessels. Herein, we evaluate the relative contribution of nitric oxide and characterize the downstream mechanisms of nitrite-induced vasorelaxation. Aortic rings were treated with pharmacological agents and exposed to hypoxia (<1% O(2)). Following pre-constriction, nitrite (10 microM final) was added to appropriate baths; isometric tension was recorded throughout. Nitrite (under hypoxic conditions at physiological pH) is capable of exerting physiological effects that cannot be completely inhibited by the inhibitor of soluble guanylate cyclase (sGC), 1H [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or a nitric oxide scavenger (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide). Simultaneous blockade of both sGC and cyclooxygenase (COX) completely inhibited the response to nitrite. With regard to the nitric oxide-dependent component, we confirm that aldehyde oxidase, but not xanthine oxidase or endothelial nitric oxide synthase, was important for the actions of nitrite in our model. Nitric oxide generated from nitrite is not exclusively responsible for the physiological actions observed in isolated hypoxic vessels. Nitrite operates via different pathways dependent on the presence or absence of endothelium to produce vasorelaxation. In intact vessels, both sGC and COX enzymes appear to be important. Irrespective of this difference in relaxation mechanism, nitrite is capable of producing the same maximum relaxation, regardless of the presence of endothelium. Having investigated possible nitrite reduction sites, we confirm that aldehyde oxidase is important for the actions of nitrite.

Catalytically Important Amino Acid Residues in Endoxylanases from Aspergillus Awamori

ABSTRACTTwo endoxylanases, Xyl-II (23 kDa) andXyl III (27 kDa), from Aspergillus awamori K-1 were modified with several specific reagents. The catalytic role of the carboxyl groups was estimated through chemical modification studies. Water-soluble carbodiimide (CMC) inactivated the xylanases rapidly and completely in a pseudo-first-order process indicating involvement of carboxyl groups in the catalysis. Treatment of the enzymes with diethyl pyrocarbonate (DEPC) resulted in modification of 1.56 histidine residues in the Xyl III (100% retention of the original enzyme activity in the presence of substrate - 0.4% xylan). Titration of the enzymes with 5.5' -dithiobis-(2-nitrobenzoic acid) (DTNB) did not result in the release of 5-mercapto-2- nitrobenzoic acid, suggesting that no free thiol groups exist in Xyl II and Xyl III. The lack of reactive thiol groups in the intact enzymes was confirmed using the specific cysteine-residue modifiers—iodoacetimide and p-chloromercuribenzoate, neither of which had any noticeable effect on xylanase activities after 60 min of incubation. N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoxylanases, suggesting that trypthophan residues are essential for the activity, since the presence of substrate protected the enzymes against inactivation.

Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.

Purification and Characterization of Two Novel β-Galactosidases from Lactobacillus reuteri

The intracellular beta-galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess beta-galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri beta-galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO ) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450 . Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO formation. HO formation in liver microsomes was inhibited by the HO radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO . Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.

Comparison of domains function between cellobiohydrolase I and endoglucanase I from Trichoderma pseudokoningii S-38 by limited proteolysis

Two homogeneous catalytic domains (CD) and cellulose-binding domains (CBD) of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma pseudokoningii S-38 are obtained from the products of limited proteolysis by papain and purified followed a series of gel filtration. A comparison of the catalytic activities and binding capacities between these two intact cellulases and their CD and CBD were made. Binding and de-sorption experiments indicate intact CBHI has stronger binding capacity on cotton fibers than others, and cannot be released from cotton fibers upon simple dilution by 20 mM NaAc, pH 4.8 buffer, while both CBDs can be completely eluted by the same buffer. Non-hydrolytic disruption of cotton fibers after adsorption of CBHI-CBD was clearly observed by scanning tunneling microscope. FT-IR spectroscopy studies demonstrate that those disruptions were driven by weakening and splitting of hydrogen bonds in cellulose. The binding capacities, as a measure of the maximum amounts of binding enzymes, and hydrolysis rates of both intact enzymes are all higher than those of the combination of their two corresponding domains in equal mole. Similarly, the effect of synergism between CBHI and EGI in hydrolysis rate of cotton fibers are also higher than those of the combination of their four domains. These results clearly suggest that the binding and catalytic functions of a cellulase molecule is mainly dependent on the coupling work of its two domains and any one domain alone is not sufficient for its full function as in an intact enzyme.

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90°C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70°C and above 70°C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.

Computer-Assisted ex vivo, Normothermic Small Bowel Perfusion

Background: In the present study, a technique for computer-assisted, normothermic, oxygenated, ex vivo, recirculating small bowel perfusion was established as a tool to investigate organ pretreatment protocols and ischemia/reperfusion phenomena. A prerequisite for the desired setup was an organ chamber for ex vivo perfusion and the use of syngeneic whole blood as perfusate. Methods: The entire small bowel was harvested from Lewis rats and perfused in an organ chamber ex vivo for at least 2 h. The temperature was kept at 37°C in a water bath. Three experimental groups were explored, characterized by different perfusion solutions. The basic perfusate consisted of syngeneic whole blood diluted with either NaCl, Krebs’ solution or Krebs’ solution and norepinephrine to a hematocrit of 30%. In addition, in each group l-glutamine was administered intraluminally. The desired perfusion pressure was 100 mm Hg which was kept constant with a computer-assisted data acquisition software, which measured on-line pressure, oxygenation, flow, temperature and pH and adjusted the pressure by changing the flow via a peristaltic pump. The viability of the preparation was tested by measuring oxygen consumption and maltose absorption, which requires intact enzymes of the mucosal brush border to break down maltose into glucose. Results: Organ perfusion in group 1 (dilution with NaCl) revealed problems such as hypersecretion into the bowel lumen, low vascular resistance and no maltose uptake. In contrast a viable organ could be demonstrated using Krebs’ solution as dilution solution. The addition of norepinephrine led to an improved perfusion over the entire perfusion period. Maltose absorption was comparable to tests conducted with native small bowel. Oxygen consumption was stable during the 2-hour perfusion period. Conclusions: The ex vivo perfusion system established enables small bowel perfusion for at least 2 h. The viability of the graft could be demonstrated. The perfusion time achieved is sufficient to study leukocyte/lymphocyte interaction with the endothelium of the graft vessels. In addition, a viable small bowel, after 2 h of ex vivo perfusion, facilitates testing of pretreatment protocols for the reduction of the immunogenicity of small bowel allografts.

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.

Differential association of the pleckstrin homology domains of phospholipases C-beta 1, C-beta 2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins.

Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-delta and PLC-gamma have been studied, the comparable domains of the beta isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-beta 1 and -beta 2 (PH-beta 1 and PH-beta 2, respectively) for lipid bilayers and G-beta gamma subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-beta gamma subunits had little affect on their membrane affinity. In contrast, binding of PLC-delta 1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-beta gamma subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-beta gamma were in the following order: PH-beta 2 >/= PH-beta 1 > PH-delta 1; the affinities of the native enzyme were as follows: PLC-beta 2 >> PLC-delta 1 > PLC-beta 1. Thus, the PH domain of PLC-beta 1 interacts with G-beta gamma in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-beta2 with G-beta gamma is comparable to that of the full-length protein and may play a key role in G-beta gamma recognition.

Effect of the chaperone-like alpha-crystallin on the refolding of lysozyme and ribonuclease A

Alpha-crystallin exhibits chaperone-like properties in preventing aggregation of proteins. We have studied the effect of alpha-crystallin on the refolding of denatured-disulfide intact and denatured-reduced lysozyme and RNase A. Alpha-crystallin does not have any effect on the refolding of both the denatured-disulfide intact enzymes. However, it inhibits the aggregation and oxidative renaturation of denatured-reduced lysozyme. Interestingly, it has no effect on the refolding of denatured-reduced RNase A. In order to probe the molecular basis of this differential behavior of alpha-crystallin towards lysozyme and RNase A, we have carried out circular dichroism and fluorescence studies on the refolding of denatured-reduced RNase A. It exhibits an extended conformation with little difference in the exposed hydrophobicity during the refolding process. We have earlier shown the presence of an aggregation-prone, refolding-competent, molten-globule-like intermediate on the refolding pathway of lysozyme. Alpha-crystallin binds to this intermediate, prevents its aggregation and inhibits its oxidative refolding. It was earlier believed that alpha-crystallin, unlike other chaperones, does not recognize intermediates on the refolding pathway but only recognizes intermediates on the unfolding pathway of proteins. Our present study clearly shows that it recognizes the refolding intermediates as well.

Reconstitution of human topoisomerase I by fragment complementation

Human topoisomerase I (topo I, 91 kDa) is composed of four major domains; the unconserved and highly charged “N-terminal” domain (24 kDa), the conserved “core” domain (54 kDa), a poorly conserved and positively charged “linker” region (5 kDa), and the highly conserved “C-terminal” domain (8 kDa) which contains the active site tyrosine at position 723. Here we demonstrate that human topo I activity can be reconstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragments that range in size from 12 kDa (linker and C-terminal domains, residues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, forming a 1:1 complex that is stable irrespective of ionic strength (0.01 to 1 M). The reconstituted enzymes are active only over a relatively narrow range of salt concentrations (25 to 200 mM KCl) as compared to the intact topo70 enzyme (missing the N-terminal domain). Under physiological conditions (150 mM KCl and 10 mM Mg2+) they are much more distributive in their mode of action than topo70. The reconstituted enzyme binds DNA with an affinity that is ∼20-fold lower than that of the intact topo70. In addition, the cleavage/religation equilibrium of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cleavage/religation equilibrium and affinity for DNA, the reconstituted and intact enzymes have identical sequence specificities for the cleavage of duplex DNA or suicide cleavage of oligonucleotide substrates.

EFFECT OF GAMMA RADIATION ON CHOLINESTERASE ENZYME

The paper studies the effect of gamma radiation on the activity of cholinesterase enzyme . The results showed that a change in the activity has occurred in the studied absorbed dose range from 10 KGy to 100 KGy . Proposals to interpret the variation of activity of irradiated cholinesterase enzyme in the studied doses are given . Moreover the effect of time after the radiolysis process on the activity of the enzyme is shown . The final conclusion suggests the replacement of gamma irradiation radiotherapy by alpha or beta radiation for local treatment of tumors to reduce the radiation noxious effects on intact cells and enzymes

The denaturation and degradation of stable enzymes at high temperatures.

Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 degrees C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation of degradation will set the upper limit of stability for enzymes.

Identification, Characterization, and Comparison of the Calmodulin-binding Domains of the Endothelial and Inducible Nitric Oxide Synthases

The calmodulin (CaM)-binding regions in bovine endothelial nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) are identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and NOS 501-532 produce a (Ca2+)-dependent, electrophoretic mobility shift of CaM on 4 M urea gels. The two peptides are also potent inhibitors of the CaM-mediated activation of neuronal nitric oxide synthase and have dissociation constants for CaM binding of 4.0 and 1.5 nM respectively. Substitution of eNOS and iNOS CaM-binding domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baculovirus/Sf9 insect cell system. Replacement of aligned NOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both (Ca2+)- and CaM-dependent. Replacement of aligned eNOS sequence with NOS 501-532 creates a functional, chimeric eNOS that is CaM-independent but that remains (Ca2+)-dependent. Specific amino acid residues critical for CaM binding by eNOS are also identified in this study as Phe-498, Lys-499, and Leu-511 in the bovine eNOS sequence.

Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain.

Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH II, have been purified from bovine brain. GDH I and GDH II were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 mM for GDH I and GDH II, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57,500 Da. Differences between the biochemical properties of GDH I and GDH II, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of alpha-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH II, isolated from bovine brain, are novel and distinct polypeptides.

Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction.

The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented.