Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts

Abstract

The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 x lw" Swiss 3T3 fibroblasts. Partial amino acid sequence data were obtained from five peptides generated by S. aureas V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic SO-87 kDa PKC substrates from both actively proliferating A43 1 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides (about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80-87 kDa PKC substrates detected in fully differentiated neural tissue.

Accelerated high‐sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge

“…Use of the minimum reagent volumes possible with the current hardware design did not cause any sequencing failures, and each aliquot of R 1 (1 5 pL) still contained a large molar excess of PITC over the peptides or proteins analyzed. A more versatile method of microcartridge operation may be obtained by the use of integrated micropathways for fluid delivery, as has been recently described for a new compact sequencer by Calaycay et al (1991). The presentation of samples on a reduced surface area was found to improve the initial yields relative to the standard system.…”

“…The revised operating programs described here have improved performance, productivity, and chemical consumption. Extensive analysis of a wide range of standard samples has revealed no inherent loss in the ability of the original sequencer to analyze both peptides and proteins with no obvious sequence-specific restrictions, and we have used the system routinely to determine the sequence of many novel peptides and proteins (Brooks et al, 1990;Kozma et al, 1990;Abo et al, 1991;Diekmann et al, 1991;Otsu et al, 1991). The increased sample turnover and sensitivity obtainable using these cycles now justifies the development of systems for multiple sample loading and very low picomole to femtomole analysis.…”

Neuropeptides as cellular growth factors: role of multiple signalling pathways

“…Furthermore, the same 80K protein is phosphorylated in cell-free systems either by activation of endogenous PKC or by addition of the purified enzyme [22,24]. Recently, this prominent PKC substrate has been purified to homogeneity from heatstable extracts of Swiss 3T3 cells [26]. The preparation is an effective substrate of PKC and contains an unusually high proportion of acidic amino acids and of alanine.…”

“…Surprisingly, the comparison of these amino acid sequences with the deduced amino acid sequence of the bovine brain substrate revealed that these proteins are closely related but are not identical (35% divergence). These PKC substrates do not exhibit significant homology to other cellular proteins [26,32]. Thus, it is well established that an increase in the phosphorylation of 80K in intact cells provides a specific marker for PKC activation.…”

“…The cDNA encoding the bovine brain protein has been cloned and sequenced [30] and specific PKC phosphorylation sites have been identified [31]. Comparison of amino acid sequences from rat brain 80K [32] and fibroblast 80K [26] demonstrated that these proteins exhibit a high degree of homology. Surprisingly, the comparison of these amino acid sequences with the deduced amino acid sequence of the bovine brain substrate revealed that these proteins are closely related but are not identical (35% divergence).…”