Alan J. Smith

Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

Abstract A method has been found which distinguishes between a size isomer family of proteins (e.g. bovine serum albumin polymers) and a charge isomer family of proteins (e.g. lactate dehydrogenase isoenzymes), utilizing disc gel electrophoresis. When the log of protein mobility relative to the dye front was plotted versus acrylamide gel concentration, size isomeric proteins gave a family of nonparallel lines extrapolating to a common point in the vicinity of 0% gel concentration; charge isomeric proteins gave a parallel family of lines. Proteins differing in both charge and size gave non-parallel lines intersecting at gel concentrations other than 0% gel concentration. The slope of such a plot is related to molecular weight. The molecular weight-slope relation was established utilizing 17 well-characterized proteins as standards. From this relation, it is possible to determine the molecular weight of a protein with an average precision of ±4%. This molecular weight method can be applied to a single protein in a mixture of proteins provided a specific detection test is available. This method should find uses in distinguishing between size and charge isomer families of proteins, for the rapid, easy, and accurate determination of protein molecular weights, and as a valuable aid in indicating the procedures to be used in enzyme purifications.

Identification of in vivo phosphorylated myosin subunits

Perrie et al. [ 1 J have shown in vitro phosphorylation of Mlz (DTNB) light chain [2] of rabbit skeletal muscle myosin. Myosin light chain Cz was phosphorylated using kinases isolated from skeletal muscle myosin and [T-~* P] ATP. Using similar conditions these investigators were not able to phosphorylate cardiac myosin. The study presented here makes a comparison between in vivo incorporation of 32P04 into each of the cardiac myosin subunits and moles of bound phosphate present in each. These phosphorylated myosin subunits were identified by both two-dimensional gel electrophoresis and isoelectrofocusing.

Microtubule-associated protein tau Identification of a novel peptide from bovine brain

The microtubule‐associated protein tau isolated from bovine brain was cleaved with CNBr and the 3 largest peptides of approx. 21, 19 and 18 kDa were obtained. Dephosphorylation of the CNBr digest of tau with alkaline phosphatase changed the electrophoretic mobility of these peptides to 19, 18 and 17 kDa. Amino acid sequencing of the total CNBr digest of tau revealed at least 3 sequences, two of which were highly homologous to previously published mouse and human tau sequences derived from cDNAs. A third amino acid sequence of 17 residues with heterogeneity at position 11 showed no homology with the cDNA‐derived tau sequences. These studies suggest that the amino acid sequences of mammalian tau predicted from their cDNAs might be incomplete.

Molecular weight studies of canine cardiac myosins

Only recently were conditions developed for determining accurately the molecular weight of myosin [ 1,2] . Conditions selective for obtaining myosin in a monomeric form were used such as, analyses of myosin at a low protein concentration ( 0.3 M KCl) [l-3] . Using these conditions the molecular weight of rabbit skeletal muscle myosin was 458 000 [l] and 468 000 [2] , molecular weights considerably lower than that reported earlier for rabbit skeletal muscle myosins [4,5] Optimal conditions for analyses of the molecular weight of myosin were used in the following studies for determination of the molecular weight of canine cardiac left and right ventricular myosins. Earlier studies on the molecular weight of myosin obtained from dog heart was described as 225 000 [6] and 758 000 [7].