Janice E. Buss

Measurement of chemical phosphate in proteins.

Publisher Summary Analytical procedures for measuring the phosphate content in proteins have suffered in general from a lack of sensitivity. This problem has required preparation of large amounts of a purified protein for phosphate analysis, a task that cannot easily be accomplished for many phosphoproteins. Such preparation is particularly difficult in cases in which the protein must be purified from tissue biopsy samples obtained for investigations of protein phosphorylation in vivo . This chapter discusses the measurement of chemical phosphate in proteins. This procedure incorporates two methods. First, the purified protein sample is ashed to convert protein-bound phosphate to inorganic phosphate. Second, the inorganic phosphate is measured after complexation of phosphomolybdate with the triphenylmethane dye, malachite green. The sensitivity of this procedure is times greater than the standard Fiske-SubbaRow procedure for measuring inorganic phosphate and measures as low as 0.2 nmol phosphate.

Sarkosyl activation of RNA polymerase activity in mitotic mouse cells.

The transition from the G2 phase of the eukaryotic cell cycle to the mitotic phase produces dramatic morphological alterations, among which are the condensation of chromatin to form chromosomes, and the disappearance of the nuclear membrane and nucleoli. These changes are accompanied by a nearly complete inhibition of cellular RNA synthesis [ 11. Recent work by Simmons et al. [2] indicates that inhibitors of protein synthesis did not prevent the resumption of RNA synthesis when mitotic cells entered the G1 phase, suggesting that RNA polymerase is present but not active during mitosis.

Properties of the polyoma virus transcription complex obtained from mouse nuclei

Abstract At a late stage of polyoma virus development in cultures of mouse 3T3 Balb/C, most of the replicated viral DNA which can be solubilized by the SDS method (1) can also be extracted as a 55S DNA-protein complex (2) by the Triton method. However, the Triton pellet fraction obtained from nuclei of infected cells was at least 50 times more active in the synthesis of polyoma-specific RNA than was the Triton supernatant upon addition of nucleoside triphosphates. This finding suggests that the active template for polyoma RNA synthesis in vivo is not the polyoma 55S complex, but rather a minor fraction of the viral DNA which is not solubilized by the Triton method. The in vitro synthesis of polyoma-specific RNA was totally sensitive to inhibition by α-amanitin. Unlike E. coli RNA polymerase, the mouse RNA polymerases were found to be resistant to inactivation by Sarkosyl.

Targeting proteins to membranes, using signal sequences for lipid modifications

Changing an existing lipid or appending a lipid to a cytosolic protein has emerged as an important technique for targeting proteins to membranes and for constitutively activating the membrane-bound protein. The potential for more precise or regulated interactions of lipidated proteins in membrane subdomains suggests that this method for membrane targeting will be of increasing usefulness.

Calcium binding to cardiac troponin and the effect of cyclic AMP-dependent protein kinase.

The contractile activity of vertebrate striated muscle appears to be regulated by the troponintropomyosin complex located in the thin filaments of the myofibril [l] . In the absence of calcium, troponin inhibits the interaction of actin and myosin. Calcium binding to troponin reverses this inhibition and allows actin and myosin to form the crossbridges which initiate muscular contraction. Troponin consists of three subunits: TN-T, a tropomyosin binding protein, TN-I, a protein which inhibits the actomyosin ATPase, and TN-C which binds calcium [2]. While cardiac and skeletal muscle troponin are functionally similar, their three subunits differ in molecular weights, amino acid composition, and column elution patterns (2). Although much information is available on calcium binding to skeletal muscle troponin, little work has been done on cardiac troponin. Using skinned frog skeletal muscle fibers and mechanically disrupted rat ventricular muscle fibers, Kerrick and Donaldson [3] concluded that similar molecular mechanism were responsible for the Ca2+-activation of the two muscle types. This was in contrast to the work of Ebashi et al. [4] who found that both the binding constant and capacity of cardiac troponin (3.4 X 10’ M-l, 1 mol Ca2’/105 g protein) were lower than that of skeletal muscle troponin. Van Eerd has compared the amino acid sequences of cardiac and skeletal muscle purified TN-C [S] and