Dexu Zhu

lactalbumin mutant acting as lysozyme

A mutant of α‐lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in α‐lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP‐(NAcGlc)3, with a KM and kcat of 0.160 ± 0.00986 mmol/L and 3.39 ± 0.0456 ×10−5 min−1, respectively, which was comparable with those of chicken lysozyme of 0.137 ± 0.0153 mmol/L and 5.25 ± 0.115 ×10‐4 min−1. By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in α‐lactalbumin could be converted into an enzyme that was 17.5‐fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond. Proteins 2001;42:17–22.

Effects of amidinopiperidine-4-Carboxylic acid 4-tert-butylphenyl ester, a specific trypsin inhibitor, on the growth of HL-60 Cells

Previous results showed that the synthetic compound amidinopiperidine‐4‐carboxylic acid 4‐tert‐butylphenyl ester (APCA‐OPhBut), a trypsin inhibitor, could specifically inhibit the activity of proteinase In and lead to growth arrest of Hela cells in early S phase. In this study, APCA‐OPhBut exhibited inhibitory effects on the growth of HL‐60 cells. Apoptotic cells were observed when the cells were cultured with APCA‐OPhBut above 50μM. Time course studies demonstrated that apoptotic cells were increased in a dose‐ and time‐dependent manner. Flowcytometric assays demonstrated that HL‐60 cells underwent slight G1 growth arrest after treatment with APCA‐OPhBut. No change of Bcl‐2 protein level was detected. The findings suggest that the intracellular trypsin‐like protease inhibited by APCA‐OPhBut not only plays a key role in DNA synthesis initiation but is also necessary for survival of certain cell lines.

Chemical conjugation of Gly‐Pro‐Arg‐Pro tetrapeptide to low molecular weight urokinase

Two low molecular weight urokinase derivatives were obtained by covalent coupling of a synthetic Gly‐Pro‐Arg‐Pro tetrapeptide to peptide A of low molecular weight urokinase and exchanging the native peptide A of low molecular weight urokinase with Gly‐Pro‐Arg‐Pro‐peptide A to obtain derivative I or direct conjugation of Gly‐Pro‐Arg‐Pro tetrapeptide to low molecular weight urokinase to obtain derivative II. In caseinolytic assay, fibrin can stimulate the two derivatives to activate plasminogen. But the two derivatives showed different kinetic behaviors. The derivative I displayed immediate onset of lysis and derivative II displayed a lag phase.

GM-CSF induces the tyrosine phosphorylation of three isoforms of shc and its association with GRB2 in TF-1 cell

This report demonstrates that GM‐CSF induces the tyrosine phosphorylation of Shc protein which is implicated in Ras activation. Three isoforms of She are ubiqitously phosphorylated induced by GM‐CSF in TF‐1, a cell line of erythroid origin. It is also shown that She is associated with the adaptor protein Grb2. The formation of Shc‐Grb2 complex may directly link tyrosine phosphorylation events to Ras activation in TF‐1 cells.

Translation initiation region plays an important role in the expression of human thrombopoietin in Escherichia coli

A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site‐specific mutagenesis based on the PCR technique. This mutant TPO gene encoded the same amino acid sequence as wild‐type TPO gene. The wild‐type TPO gene was expressed in E. coli with very low efficiency. The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E. coli, which was much higher than the wild‐type gene. The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding.

A low molecular weight urokinase derivative with enhanced fibrin affinity.

A Gly‐Pro‐Arg‐Pro tetrapeptide, homologous to amino‐terminal segment of the human fibrin α chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had two‐fold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9‐fold higher than that of low molecular weight urokinase.

A urokinase mutant with high fibrin-selectivity

BECAUSE of lacking fibrin selectivity two chain urokinase-type plasminogen activator(tcu-PA),which has been used as the clinical thrombolytic agent,may lead to bleeding by a remarkabledepletion of fibrinogen in plasma when injected in large doses,thus its clinical application islimited.Single chain urokinase-type plasminogen activator(scu-PA)is the precursor of tcu-

Enhancement of in vitro renaturation of recombinant human pro‐urokinase by ampicillin

Ampicillin was used in vitro renaturation process of recombinant human prourokinase and it can obviously improve the yield of the renaturation, although not as well as arginine. When ampicillin was used together with arginine or lysine, it decreased the yield of the renaturation comparing with the yield when arginine or lysine used alone.

Mutation of Arg154 to Gly154 in urokinase augments its fibrin‐specificity

Rscu‐PA and its mutant constructed by in vitro site specific mutagenesis of Arg154 in rscu‐PA to Gly154 (mscu‐PA) were both expressed in Escherichia coli. After in vitro denaturation and renaturation, the rscu‐PA and mscu‐PA were purified to homogeneity by Zn2+ selective precipitation, anti‐u‐PA IgG‐sepharose CL 4B affinity chromatography. After activation by plasmin, the kinetic constants for the resultant mtcu‐PA against synthetic substrate S2444 hydrolysis were found to be essentially identical to rtcu‐PA, suggesting that no impairment had been exerted on the catalytic active site of mtcu‐PA. However, both 125I‐fibrin plasma‐clot lysis and fibrinogenolysis showed that mtcu‐PA possessed a higher fibrinolytic activity but hardly any degradation of fibrinogen in plasma compared to rtcu‐PA and rscu‐PA. It was concluded that the substitution of Arg154 by Gly154 in tcu‐PA promoted the fibrin‐specificity of urokinase.