Kinetic substrate quantification by fitting the enzyme reaction curve to the integrated Michaelis-Menten equation.

The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis-Menten equation was investigated by both simulation and preliminary experimentation. For simulation, product absorptivity epsilon was 3.00 mmol(-1) L cm(-1) and K(m) was 0.10 mmol L(-1), and uniform absorbance error sigma was randomly inserted into the error-free reaction curve of product absorbance A(i) versus reaction time t(i) calculated according to the integrated Michaelis-Menten equation. The experimental reaction curve of arylesterase acting on phenyl acetate was monitored by phenol absorbance at 270 nm. Maximal product absorbance A(m) was predicted by nonlinear fitting of the reaction curve to Eq. (1) with K(m) as constant. There were unique A(m) for best fitting of both the simulated and experimental reaction curves. Neither the error in reaction origin nor the variation of enzyme activity changed the background-corrected value of A(m). But the range of data under analysis, the background absorbance, and absorbance error sigma had an effect. By simulation, A(m) from 0.150 to 3.600 was predicted with reliability and linear response to substrate concentration when there was 80% consumption of substrate at sigma of 0.001. Restriction of absorbance to 0.700 enabled A(m) up to 1.800 to be predicted at sigma of 0.001. Detection limit reached A(m) of 0.090 at sigma of 0.001. By experimentation, the reproducibility was 4.6% at substrate concentration twice the K(m), and A(m) linearly responded to phenyl acetate with consistent absorptivity for phenol, and upper limit about twice the maximum of experimental absorbance. These results supported the reliability of this new kinetic method for enzymatic analysis with enhanced upper limit and precision.

[Expression and identification of recombinant arresten in Pichia pastoris].

Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.

[Preparation and identification of the monoclonal antibody against human endostatin].

AIM: To prepare monoclonal antibody against human endostatin for the in depth study on the mechanism of anti-tumor effect of endostatin. METHODS: Monoclonal antibody specific for endostatin was prepared using hybridoma technique and screened with ELISA. Ascites fluids were produced in BALB/c mice following in sequentical intraperitoneal injection of pristine and hybridoma cells. The mAb was purified by affinify chromatography with protein A sepharose CL-4B. RESULTS: One hybridoma cell line 4E7 was established, which could produce mAb against human endostatin. The titers of mAb 4E7 in culture supernatant and ascites fluid were 1:128-1:516 and 1:10(-4)-1:10(-6), respectively. The mAb 4E7 belonged to IgG1 (lambda type). Western blot analysis showed that the mAb 4E7 could react to the human endostatin expressed by yeast and E.coli, but it had no cross reaction to other cytokines such as bFGF. CONCLUSION: The mAb 4E7 is able to react specifically with human endostatin and can be used for further investigation.

Assay of Adenylyl Cyclase Activity by Ion-exchange High-performance Liquid Chromatography.

Adenylyl cyclase(AC)activity was determined by directly quantifying the product cAMP with ion-exchange HPLC. When AC reaction was terminated, the reaction mixture was kept at 0 degrees for 2 h to remove residual ATP utilizing the action of ATP-hydrolyzing enzymes existed in the crude AC preparation of membrane pellet. Papaverine was removed by the dichloromethane extraction, and cAMP was quantified by chromatography on a WAX-1 HPLC column with baseline-resolution. It was found that cAMP was linearly formed within 10 min of the reaction, and the amount of cAMP formed was proportional to the amount of enzyme. Mouse brain AC activity determined by this method was 42 nmol/min per gram of protein, similar to that obtained by the classical [(3)H] labeled ATP method. This ion-exchange HPLC was more rapid and convenient.