Low Molecular Weight Substrates Research Articles

Acylation of amino functions of proteins with monomethoxypoly (ethylene glycol)-N-succinimide carbonate

Monomethoxypoly(ethylene glycol)-N-succinimide carbonate (SC-PEG) was used to prepare PEG-lysozyme, PEG-papaya proteinase III, PEG-catalase, and PEG-lactoperoxidase conjugates. SC-PEG produced extensively modified enzymes under mild conditions (pH 7.0; 25°C) within a couple of hours. PEG-enzyme conjugates showed equal or even greater specific activity provided that low-molecularweight substrates were used to evaluate the biological activities. However, papaya proteinase III and lysozyme lost their proteolytic and bacteriolytic activities, respectively, on conjugation with PEG. This was most probably because of steric factors, since no drastic conformational changes could be detected after conjugation of these enzymes with PEG chains. Unlike these enzymes, the secondary structures of the two hemoproteins were somewhat affected by the covalent attachment of PEG chains as shown by FTIR experiments. These results confirmed the potential usefulness of SC-PEG, for which a novel route of synthesis making use ofN,N′-disuccinimidyl carbonate was described.

Substrate specificity of collagenolytic proteases from the king crab Paralithodes camtschatica

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro- d-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.

The gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IXa beta. Effects on the serine protease module and factor X activation.

Blood coagulation factors IX and X are two serine proteases with a similar modular structure. The non-catalytic part of each protein consists of a gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. We have now found that the NH2-terminal EGF-like module of both factors IX and X inhibits factor Xa formation in a Gla-independent manner, both in the presence and absence of phospholipid and the cofactor, factor VIIIa. In contrast, the COOH-terminal EGF-like module has no such effect. Our data indicate that the NH2-terminal EGF-like module of factor IXa beta interacts either with the corresponding module or with the serine protease module in the substrate, factor X, without affecting the hydrolysis of low molecular weight substrates. Using antibodies as structural probes, we found that Ca2+ binding to the Gla module of factor IXa beta induces a conformational transition in the serine protease module. No evidence was found for a direct interaction between the Gla module and factor VIIIa. We therefore propose that the Gla module in factor IXa beta is indirectly involved in the cofactor interaction, in that Ca2+ binding to sites in this module induces a conformation in the serine protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca2+ binding site in the serine protease module (apparent Kd of approximately 120 microM) that also might influence its conformation. Antibodies against the EGF-like modules of factor IX were used to probe Ca2+ binding to these modules in intact and in Gla-domainless factor IXa beta. The data indicate a Ca2+ binding site with an apparent Kd of approximately 50 microM in the NH2-terminal EGF-like module of both factor IX species.

Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane

The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaccharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of α-CD to β-CD and MOS or β-CD to α-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization =2–6) was disproportionated with a conversion ratio exceeding 70% by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.

Altered relation between granulocyte elastase and alpha-2-macroglobulin in gingival crevicular fluid from sites with periodontal destruction.

Granulocyte elastase activity and alpha-2-macroglobulin (alpha-2-MG) were studied in the gingival crevicular fluid (GCF) from 3 categories of sites in 6 patients with gingivitis and 6 patients with periodontitis. 6 inflamed sites in each gingivitis patient were sampled on paper strips and 12 sites, 6 with and 6 without attachment loss and periodontal pockets, were selected in each periodontitis patient. To avoid the influence of increase GCF volume from deep pockets, the elastase activity and the alpha-2-MG were calculated per microliters of GCF. The proteolytic activity of elastase was measured with a low molecular weight substrate and the antiprotease, alpha-2-MG, with ELISA. The measured activity could be ascribed to elastase that had been released into the gingival tissues and into the GCF prior to sampling. In the periodontitis patients, the sites with tissue destruction had a significantly higher elastase activity per site and per microliters GCF and a significantly lower alpha-2-MG per microliters than the 2 other categories of sites without tissue destruction. The destructive inflammation seems to be associated with increased release of elastase, either from more numerous or from more active granulocytes and with an increased proteolytic consumption of the inhibitor accompanied by the fast elimination of the protease-inhibitor-complex. In conclusion, the study shows a strong relationship between elastase activity and tissue destruction, a finding that supports the pathogenic theory of an involvement of granulocytes and their proteolytic enzymes in the mechanism of periodontal destruction.

Expression, purification, and characterization of SH2-containing protein tyrosine phosphatase, SH-PTP2.

A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.

Correction - Enzymatic Activity of Chymotrypsin and Its Poly(Ethylene Glycol) Conjugates Toward Low and High Molecular Weight Substrates

Correction - Enzymatic Activity of Chymotrypsin and Its Poly(Ethylene Glycol) Conjugates Toward Low and High Molecular Weight Substrates

Enzymatic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates.

Native chymotrypsin and its polyethylene glycol (PEG) conjugates, obtained using the succinimidyl carbonate of methoxy-PEG (SC-PEG) as the amino group modifying reagent, were tested for their activity toward several low and high molecular weight substrates. Tripeptide and tetrapeptide p-nitroanilides either as N alpha-benzyloxycarbonyl derivatives or attached via their N-terminals to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers or to PEG, as well as the proteins albumin and azoalbumin, were used as substrates. Extensively modified chymotrypsin bearing on average of 14 PEG chains per chymotrypsin molecule (PEG14CHT) and a moderately modified one containing 10 PEG chains (PEG10CHT) both degraded the low molecular weight p-nitroanilides at rates comparable to, and in the case of PEG10CHT, greater than, the rates exhibited by the native enzyme. Synthetic high molecular weight substrates and azoalbumin were also degraded by the PEG-modified enzymes. However, rates of such enzymatic reactions were lower for the PEG-enzymes than for the native one. Native albumin, as compared to azoalbumin, resisted degradation by both PEGCHTs yet was readily digested by the native chymotrypsin. The results obtained indicate that substrate-size-dependent specificity of PEG-modified enzymes cannot be explained solely by steric hindrance considerations. The decreased activity of PEG-enzymes toward protein substrates is consistent with the well-documented ability of PEG to exclude proteins from its surroundings and with the influence of protein unfolding on the susceptibility to degradation.

Functional effects of kringle 2 glycosylation in a hybrid plasminogen activator

uK2t-PA is a hybrid plasminogen activator in which the epidermal growth factor-like domain of the urokinase-type plasminogen activator precedes the kringle 2 and catalytic domains of tissue-type plasminogen activator. The molecules are expressed in Chinese hamster ovary cells in two variant forms, a type II form in which only the protease domain is glycosylated, and a type I form in which both the kringle 2 and the protease domains carry N-acetyllactosamine type glycans. The two forms differed slightly in their affinity for fibrin and fibrinogen, which allowed their separation, but the stimulation of plasminogen activation of the type II form by fibrin was up to eight-fold lower than that of the type II form. The sensitivity to fibrin could be restored by treatment of the type I form with N-glycanase or sialidase. Enzymatic activity vs low molecular weight substrates was not influenced by the glycosylation of kringle 2.

Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis.

The specificities of 15 cellulolytic enzymes have been examined using chromophoric glycosides derived from D-glucose, cellobiose, higher cellooligosaccharides, lactose, D-xylose, and beta-(1,4)-xylobiose. Coinciding with a classification based on hydrophobic cluster analysis of amino acid sequences, six families each showing a characteristic specificity pattern were observed. Furthermore, in these cases where the anomeric forms of reaction products were determined, results seem to indicate conservation of intrinsic reaction mechanism (single or double displacement) within each family. On the other hand, the low molecular weight substrates do not discriminate exo- from endocellulases. This functional differentiation is speculated to originate from the presence, in exoenzymes, of a tunnel-shaped active site formed by extra loops in their structure.

Granulocyte elastase in gingival crevicular fluid. A possible discriminator between gingivitis and periodontitis.

The granulocyte elastase activity and the immuno-reactive (antigenic) granulocyte elastase of gingival crevicular fluid (GCF) were studied in 16 periodontitis patients and in 10 gingivitis patients. The elastase activity was measured with a low molecular weight substrate specific for granulocyte elastase. The antigenic elastase was determined with specific antibodies against granulocyte elastase. Intracrevicular sampling of GCF with paper strips for 30 s seemed to provide representative values of elastase. The elastase activity correlated with probing depth and attachment loss and appeared to be a measure of the degree of tissue destruction. Antigenic elastase represents the number of granulocytes in GCF and should thus be related to the degree of inflammation. The periodontitis patients and the gingivitis patients both had a similar degree of inflammation as measured by antigenic elastase per microliter GCF and gingival index. The elastase activity per microliter GCF, however, was higher in the periodontitis group. Elevated granulocyte elastase activity in GCF seems to be independent of inflammation and could thus be an indicator of patients at risk for periodontitis.

Proteinase inhibitory activity in the plasma of a mollusc: Evidence for the presence of α-macroglobulin in Biomphalaria glabrata

1. 1. A methylamine-sensitive inhibitor was present in the plasma of B. glabrata. 2. 2. This inhibitor decreased trypsin activity against a protein substrate, however trypsin retained activity against a low molecular weight substrate in the presence of the inhibitor. 3. 3. Snail plasma protected trypsin from inhbition by soybean trypsin inhibitor. 4. 4. The results give evidence for an α-macroglobulin proteinase inhibitor in the plasma of this gastropod mollusc.

Ethanol-extractable substrate pools and the incorporation of thymidine, L-leucine, and other substrates by bacterioplankton

Bacterioplankton productivity measurements based on [methyl-3H]-thymidine (TdR) or L-[3,4,5-3H]leucine (L-leu) incorporation typically depend on cold trichloroacetic acid (TCA) precipitation to separate 3H uptake from incorporation. An additional rinse with cold 80% ethanol (EtOH) removed an average of 22 (L-leu) and 32% (TdR) of 3H "incorporated" by San Francisco Bay samples and decreased the between-duplicate difference by a factor of 3.5. Similar results were obtained with samples from Tomales Bay, Calif., and Palmer Station, Antarctica. Varying amounts of cold TCA insoluble radiolabel from six other substrates were removed by the EtOH rinse. Regression analysis showed relationships between the effect of the EtOH rinse and a group of environmental variables and derived parameters. The percentage of 3H removed was generally independent of filter type; however, there were often large differences in the amount of 3H retained by Millipore versus Nuclepore or Poretics filters. The results strongly suggest that an EtOH rinse or other organic extraction should be included in protocols to determine incorporation of radiolabeled substrates into macromolecules. Furthermore, sequestering low molecular weight substrates in some sort of lipid-bound pool may represent a general storage mechanism employed by bacterioplankton. Key words: bacterioplankton, production, San Francisco Bay, filtration, incorporation.

Purification and characterization of a Coffea canephora α-D-galactosidase isozyme

Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids. The α-D-galactosidase from Coffea canephora is an important exoglycosidase which degrades the human blood group B epitope. Although multiple isozymes have been described, they have never been demonstrably purified and thoroughly characterized. We have developed a technique to purify an isozyme to homogeneity. The isolated enzyme has a molecular weight of 36.7 kDa by SDS PAGE and 34.0 kDa by gel filtration. The isozyme is highly selective for α-D-galactosides and inactive against other low molecular weight substrates. It hydrolyzes the the terminal α-D-galactosyl residue from the blood group B epitope. Protease activity is below detectable limits. The isozyme has a broad pH optima at 6.3, a pl of 7.03, is unaffected by ionic strength, and is stable at 4° C.

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP)

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN′) and uridine and cytidine 2′, 3′-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the ‘non-secretory’ liver-type enzymes rather than to the ‘secretory’ pancreatic-type RNases.

A Continuous Cell-Free Protein Synthesis System for Coupled Transcription-Translation1

A continuous cell-free protein synthesis system [Spirin et al. (1988) Science 242, 1162-1164] was modified so as to be suitable for coupled transcription-translation, a process useful for obtaining products of cloned genes or cDNAs. A reaction chamber equipped with an ultrafiltration membrane was newly designed and an HPLC pump was used to supply a low molecular weight substrate solution at a constant rate to the viscous reaction mixture in the chamber. By using an Escherichia coli S30 extract in this modified flow system (1 ml), coupled transcription-translation could be continuously performed for 17 h, the synthesized chloramphenicol acetyltransferase (congruent to 0.1 mg) being subsequently eluted through the chamber's membrane and then purified.

The 1,4-β- d-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei

A physico-chemical and structural characterization of three 1,4-β- d-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.

Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.

Decreased specific anti-elastase activity in the uninvolved skin of patients with psoriasis

Inhibitory activities against elastase, chymotrypsin and trypsin were studied in the fluid from experimentally developed suction blisters in the uninvolved skin of patients with psoriasis. These activities determined by spectrophotometry of specific synthetic low molecular weight substrates were compared with respective antiproteinase activities in sera of 32 patients with psoriatic lesions, ten patients in remission, and ten healthy volunteers. A marked reduction (29.2%) in the specific elastase inhibitory activity of blister fluid was found in patients with psoriasis when compared with normal subjects (p less than 0.05), since neither chymotrypsin nor trypsin inhibitory activities were altered. This reduction was despite about a 30% increase in the elastase inhibitory activity in the sera of these patients, which was related presumably to their increased activity of alpha 1-proteinase inhibitor, the main serum antiserine proteinase inhibitor. A decreased blister fluid:serum elastase inhibition ratio was shown in a large majority of patients with psoriasis, even in symptomless patients. The deficiency in specific elastase inhibitory activity of suction blister fluid was predominantly associated with early onset of psoriasis, guttate lesions and inactive lesions, skin involvement less than 20% of body surface, duration of relapse shorter than 2 months, and frequent relapses. These data indicate that the uninvolved skin of patients with psoriasis contains low concentrations of specific elastase tissue inhibitor, which deficiency might result in an excessive in vivo hydrolytic activity of neutrophil elastase released from migrating cells in the psoriatic skin.

A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.