Assay and Purification

Because of MLCK s central role in smooth muscle contraction and various nonmuscle cell processes, many investigations have focused on the biochemical and molecular properties of purified MLCK. Purification schemes for MLCK from mammalian smooth muscle tissues generally involve modifications of the procedure originally described for avian smooth muscle MLCK (Adelstein and Klee, 1981). The kinase has been purified from many types of mammalian smooth muscles, including trachea, aorta, myometrium, and stomach (Stull et al., 1986). 

The following procedure describes a purification scheme for MLCK from bovine tracheal smooth muscle (Stull et al., 1990). All purification procedures 

1 mM phenylmethylsulfonyl fluoride, 0.1 mM N -p-tosyl-L-lysine chloromethyl ketone, 0.1 nM L-l-tosyl-amido-2-phenylethyl chloromethyl ketone, 8 mg/liter lima bean trypsin inhibitor, and 10 pM leupeptin at pH 

1 mM dithiothreitol at pH 6.8. The dialyzed sample is applied to a hydroxylapatite column (1x4 cm). After washing the column in the same buffer, the kinase is eluted with a 10 to 300 mM potassium phosphate gradient containing 1 mM dithiothreitol at pH 6.8 in a total volume of 120 ml. The fractions containing MLCK activity are brought to 10% glycerol and stored at -60°C. 

BIOCHEMICAL AND MOLECULAR PROPERTIES OF MYOSIN LIGHT CHAIN KINASE  

---

Ali, M. Brownstone, Y.S. A study of phosphoglycerate kinase in human erythrocytes. 1. Enzyme isolation, purification and assay. Biochim. Biophys. Acta, 445, 74-88 (1976)... 

Methods of Enzymatic Analysis, H. Bergmeyer, Editor. Contains methods for enzyme purification and assay, in several volumes. 

G Zhao, TI Meier, WK Yeh. Penicillin-binding proteins as antimicrobial targets expression, purification, and assay technologies. In HA Kirst, WK Yeh, MJZmijew-ski, Jr., eds. Enzyme Technologies for Pharmaceutical and Biotechnological Applications. New York Marcel Dekker, 2001, pp. 263-287. 

Penicillin-Binding Proteins as Antimicrobial Targets Expression, Purification, and Assay Technologies... 

Purification and Assay Development for Human Rhinovirus Proteases... 

The active 2A and 3C proteases from different HRV serotypes have been overproduced in bacterial cells and purified by several groups [18,28-42], The availability of large quantities of the recombinant viral proteases has greatly facilitated their biochemical characterization, assay development, and efforts to identify specific inhibitors against these enzymes. We have worked on the purification and assay development for the two proteases from HRV serotype-14 (HRV 14) as part of our efforts in antiviral development. The experimental procedures described below may provide a general approach toward the purification and development of high-throughput assays for the 2A and 3C proteases of other HRV serotypes as well as other members of the picomaviral family. 

In the early days of biotechnology product development, the focus was on quality issues [4] or process-related impurities.The concerns at that time were for carryover of other cellular proteins and DNA and for contamination with endotoxins, chemicals, and viruses. Of course, these concerns still exist, but methods for purification and assays for evaluation of clearance have alleviated the need for the safety assessment scientist to focus on contaminants instead they are now asked to focus on the pharmacological activity of the molecules. An ICH guidance (Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) addresses the specific issues related to the manufacturing process [6], Other product-related issues such as impurities do need to be considered by the safety assessment scientist, for... 

INITIAL PURIFICATION AND ASSAY OF ACTIVITIES IN CEU--FREE LYSATES 105... 

W13. Williams, W. L, and Ellenbogen, L., Purification and assay of intrinsic factor. Vitamin Bjo und Intrinsic Factor, 1. Europ. Symp., Hamburg, 1956 pp. 206-213. Enke, Stuttgart, 1957. 

Dillon, ST and Feig, LA (1995) Purification and assay of recombinant C3 transferase. In Methods in Enzymology (Balch, WE, Der, CJ, Hall, A eds) Vol 256, ppl74—84, San Diego, Academic Press. 

Alouf JE, Geoffroy C (1988) Production, purification and assay of streptolysin O. In Microbial Toxins Tools in Enzymology. Methods in Enzymology 165 (Harshman S, ed) pp 52-59, Academic Press, San Diego. 

A. B. Theibert G. D. Prestwich T. R. Jackson L. P. Hammonds-Odie, The Purification and Assay of Inositide Binding Proteins. In Signalling by Inositides, S. B. Shears, Ed. Oxford University Press London, 1997 p 117. 

Introduction.—This Report covers the literature published up to approximately the end of September, 1981. Few new carotenoid structures have been reported. The main advances in carotenoid chemistry have been in the stereospecific synthesis of carotenoids with chiral end-groups. Current interest in the possible use of retinoids in cancer chemotherapy has prompted the preparation of a considerable number of retinoic acid analogues. There has been no major new development in the use of physical methods but h.p.l.c. becomes more and more the method of choice for carotenoid separation, purification, and assay, and the increasing number of papers on resonance Raman spectroscopy emphasizes the potential value of this technique in the carotenoid field. 

---