Jeffrey R. Davies

Resolution of Ca2+—calmodulin-activated protein kinase from wheat germ

A soluble Ca2+‐ and Ca2+—calmodulin‐activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca2+‐dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3‐fold and up to 16‐fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca2+‐dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca2+‐dependent fashion. This type of Ca2+‐activated protein kinase may be involved in stimulus—response coupling in plants.

Purification of some tricarboxylic acid cycle enzymes from beef heart using affinity elution chromatography

Abstract Applying the principles of affinity elution chromatography, eight different mitochondrial enzymes of, or associated with, the tricarboxylic acid cycle have been purified from beef heart. Conditions were found for adsorbing each enzyme to CM-cellulose, and eluting each with its substrate(s). After a gel filtration step, and in some cases use of an affinity adsorbent, most of these preparations had specific activities comparable to the highest previously reported.

Gamma globulin-derived standards for the determination of molecular weights, transfer, and immunodetection efficiencies in protein blotting procedures

Molecular weight markers which are detectable using labeled antispecies antibodies or labeled Protein A have been prepared for use as standards on protein blots. The standards were prepared by the controlled reduction followed by subsequent alkylation of gamma globulin. Separate sets of standards were prepared using gamma globulins derived from human, mouse, rabbit, and sheep species. Standards were also prepared using monoclonal-derived gamma globulins from human myeloma fluid and mouse ascites fluid. Standards produced from monoclonal-derived gamma globulins produced very sharp bands on sodium dodecyl sulfate-polyacrylamide gels and proved to be excellent standards for this technique alone. However, the markers were uniquely suitable for use as standards in protein blotting procedures because their detection was achieved by the procedure used to detect the transferred antigen(s). The detection of immunoglobulin G (IgG)-derived standards on protein blots from all the species listed above was demonstrated using appropriate horseradish peroxidase (HRP)-conjugated antispecies antibodies. The use of other detection systems (biotin-labeled antibody and subsequent detection with HRP-steptavidin, HRP-Protein A) was also validated with human IgG-derived standards. Furthermore, the standards were shown to be suitable for use on both nitrocellulose and cationized nylon-based supports and could be used when adjacent samples were run under reducing conditions. Hence the gamma globulin-derived standards serve as both a control to check the adequacy of transfer and immunodetection systems and as markers which enable the molecular weights of detected antigens to be calculated.