The selective deletion of a discrete surface-exposed epitope (residues 254 -262; 250-loop) in the ␤ domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK del254 -262 ). A kinetic analysis of SK del254 -262 revealed that its low HPG activator activity arose from a 5-6-fold increase in K m for HPG as substrate, with little alteration in k cat rates. This increase in the K m for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK del254 -262 . The involvement of kringles was further indicated by a hypersusceptibility of the SK del254 -262 ⅐ plasmin activator complex to ⑀-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK ⅐ plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK del254 -262 ⅐ HPG was reduced by about 3-fold in comparison with that of nSK⅐HPG. Overall, these observations identify the 250 loop in the ␤ domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK⅐HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process. Streptokinase (SK),1 a bacterial protein secreted by the Lancefield Group C ␤-hemolytic streptococci, is widely used as a thrombolytic agent in the treatment of various circulatory disorders, including myocardial infarction (1). Unlike other human plasminogen (HPG) activators, like tissue plasminogen activator and urokinase, SK does not possess any intrinsic enzymatic activity. Instead, SK forms an equimolar, stoichiometric complex with "partner" HPG or plasmin (HPN), which then catalytically activates free "substrate" molecules of HPG to HPN by selective cleavage of the Arg 561 -Val 562 peptide bond (2, 3). It is believed that consequent to the initial SK⅐HPG complexation, there is a structural rearrangement within the complex, and even before any proteolytic cleavage takes place, an active center within the HPG moiety capable of undergoing acylation is formed (3). This activated complex is rapidly transformed into an SK⅐HPN complex and develops an HPG activator activity. Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK⅐HPN displays a very narrow substrate specificity (4). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the "cofactor" SK, with exclusiv...

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

Although several recent studies employing various truncated fragments of streptokinase (SK) have demonstrated that the high-affinity interactions of this protein with human plasminogen (HPG) to form the activator complex (SK-HPG) are located in the central region of SK, the exact location and nature of such HPG interacting site@) is still unclear. In order to locate the "core" HPG binding ability in SK, we focused on the primary structure of a tryptic fragment of SK derived from the central region (SK143-293) that could bind as well as activate HPG, albeit at reduced levels in comparison to the activity of the native, full-length protein. Because this fragment was refractory to further controlled proteolysis, we took recourse to a synthetic peptide approach wherein the HPG interacting properties of 16 overlapping 20-mer peptides derived from this region of SK were examined systematically. Only four peptides from this set, viz., SK234-253, SK254-273, SK274-293, and SK263-282, together representing the contiguous sequence SK234-293, displayed HPG binding ability. This was established by a specific HPG-binding ELISA as well as by dot blot assay using '251-labeled HPG. These results showed that the minimal sequence with HPG binding function resided between residues 234 and 293. None of the synthetic SK peptides was found to activate HPG, either individually or in combination, but, in competition experiments where each of the peptides was added prior to complex formation between SK and HPG, three of the HPG binding peptides (SK234-253, SK254-273, and SK274-293) inhibited strongly the generation of a functional activator complex by SK and HPG. This indicated that residues 234-293 in SK participate directly in intermolecular contact formation with HPG during the formation of the 1: 1 SK-HPG complex. Two of the three peptides (SK234-253 and SK274-293), apart from interfering in SK-HPG complex formation, also showed inhibition of the amidolytic activity of free HPN by increasing the K, by approximately fivefold. A similar increase in K, for amidolysis by HPN as a result of complexation with SK has been interpreted previously to arise from the steric hinderance at or near the active site due to the binding of SK in this region. Thus, our results suggest that SK234-253 and SK274-293 also, like SK, bound close to the active site of HPN, an event that was reflected in the observed alteration in its substrate accessibility. By contrast, whereas the intervening peptide (SK254-273) could not inhibit amidolysis by free HPN, it showed a marked inhibition of the activation of "substrate" PC (human or bovine plasminogen) by activator complex, indicating that this particular region is intimately involved in interaction of the SK-HPG activator complex with substrate plasminogen during the catalytic cycle. This finding provides a rational explanation for one of the most intriguing aspects of SK action, Le., the ability of the SK-HPG complex to catalyze selectively the activation of substrate molecules of PC to PN, whereas free...

The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2-and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:l SK-HPG activator complex was tested. Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51. However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK. Although SK16-5 1 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle. On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK acti...

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