PubMed · 5179224
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T Iizuka. 1966. [Insulin].. https://pubmed.ncbi.nlm.nih.gov/5179224/
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A novel method for studying the ionization step of matrix-assisted laser desorption/ionization (MALDI) is demonstrated. A 193-nm pulse from an ArF excimer laser is used to photodissociate a portion of a plume of insulin ions generated by MALDI. Laser photodissociation (LPD) creates a "hole", i.e., a negative spike in the insulin peak in the time-of-flight (TOF) mass spectrum. The position of the hole in the mass spectrum provides useful measurements of the characteristics (position, time, and velocity) of insulin ions shortly after their creation. Although the performance of the method can be further improved, the data obtained could be used to refine our current understanding of MALDI and to improve the resolution of MALDI-TOFMS.
In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR beta-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.
The determination of deshexapeptide (B25-B30) insulin (DHI) was divided into two steps. At the first step, the rough structure model of DHI molecule was determined by using the molecular replacement method associated with the molecular close-packing method at 0.30 nm resolution based on the reflection data collected on four-cycle diffractometer. At the second step, the DHI model was adjusted and refined at 0.25 nm resolution based on the data collected on Area Detector. 40 water molecules were determined during the refinement, the final R-factor is 0.185 with R.M.S. deviation of 0.002 nm for bond lengths and 1.9 degrees for bond angles. The differences in conformation and function of DHI with other insulin analogues were compared and discussed.