Lonnie D. Adams

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.

The Lipolytic System in Adipose Tissue of Toronto-KK and C57BL/KsJ Diabetic Mice. Adenylate Cyclase, Phosphodiesterase and Protein Kinase Activities

SummaryEpinephrine-stimulated lipolysis in epididymal fat pads of T-KK mice is low, compared to C57BL/6J controls, at 10–12 weeks and 8–9 months of age. Lipolysis in isolated fat cells is similarly unresponsive to epinephrine in 9–12 week old T-KK anddb mice, but cyclic AMP phosphodiesterase activity is normal. Basal adenyl cyclase activity in ghosts prepared from isolated fat cells of T-KK mice is elevated but epinephrine-stimulated activity is low compared to controls. Total cyclase activity, elicited by NaF, is higher in T-KK mice. Adenyl cyclase activity in particulate fractions prepared from fat pad homogenates ofdb mice is low compared to control mice when expressed as total activity per fat pad. Unstimulated and cyclic AMP-stimulated protein kinase activities in fat pad homogenates are normal in T-KK mice, but indb mice unstimulated activity is depressed while stimulated activity is elevated.

Two‐Dimensional Gel Electrophoresis Using the ISO‐DALT System

Two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) are combined in this unit to provide much greater resolution than either of the individual procedures. Solubilized proteins are first separated according to their isoelectric point by isoelectric focusing in a tube gel. This first dimension gel is then applied to the top of an SDS‐polyacrylamide slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are further separated on the basis of their molecular size. The ISO‐DALT system was specifically designed for running multiple high‐resolution two‐dimensional gels at one time.

Rapid modulation of a 64 K Dalton fibroblast protein: a PDGF mediated early cellular event

The results presented here reveal a novel platelet derived growth factor (PDGF) mediated early cellular event. Treatment of growth arrested Balb/c3T3 fibroblasts with PDGF induces a specific and rapid modulation of a 64,000 Dalton (64 KD) protein preexisting in quiescent cells. The kinetics of 64 KD protein modulation indicate that, temporally, this PDGF mediated step lies between the membrane associated immediate events such as receptor autophosphorylation or ion mobilization and the earliest known transcriptional event, the activation of the proto-oncogene c-fos.

Two-Dimensional Gel Electrophoresis of Polypeptides

We report a method for separating proteins and peptides in the range Mr 2,000 to Mr 200,000 by SDS-polyacrylamide gel electrophoresis without urea or a stacking gel. This system was developed as an alternative to the Swank and Munkres (1) and Kyte and Rodriguez (2) procedures previously used in our laboratory because they were limited to penetration of protein and peptides less than Mr 50,000 and require urea. Other gel systems which cover an extended molecular weight range have been reported (3,4) but also require urea. The essential features of this gel include gradients of acrylamide, N,N-methylenebisacrylamide (bis), and glycerol in a trisphosphate buffer system. A two-dimensional separation of an E. coli extract first run on a conventional isoelectric focusing tube gel followed by our SDS-PAGE gel is given as an example of its utility in the laboratory.