|Description||Inhibitor 2, PP1 specific|
|Overview||expressed in E. coli.|
|Application||Inhibits PP1 from many sources (human,
rabbit, yeast). For use in Kinase Assays.
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||1 year at -70°C|
|Material Size||100 µg|
|Inhibitor 2 (specific inhibitor of PP1) - 2187210||2187210|
|Inhibitor 2 (specific inhibitor of PP1) - 2277839||2277839|
|Inhibitor 2 - 16429||16429|
|Inhibitor 2 - 17622||17622|
|Inhibitor 2 - 18478||18478|
|Inhibitor 2 - 2048885||2048885|
|Inhibitor 2 - 26521||26521|
|Inhibitor 2 - DAM1606769||DAM1606769|
|Inhibitor 2 - DAM1791371||DAM1791371|
|Inhibitor 2 - JBC1863285||JBC1863285|
|Reference overview||Pub Med ID|
|Cloning of the complete coding region for human protein phosphatase inhibitor 2 using the two hybrid system and expression of inhibitor 2 in E. coli. |
Helps, N R, et al.
FEBS Lett., 340: 93-8 (1994) 1994
The yeast two hybrid system has been employed to identify cDNAs encoding proteins which interact with the gamma 1 isoform of human protein phosphatase 1. Here we report the isolation of cDNA encoding human protein phosphatase inhibitor. The deduced human sequence of 205 amino acids shows 92% identity to inhibitor 2 from rabbit. Human inhibitor 2 was expressed in E. coli and purified to homogeneity. The expressed human protein inhibited both native and bacterially expressed PP1, with the same Ki (1 nM) as inhibitor 2 purified from skeletal muscle. A gene or pseudogene for inhibitor 2 may be present near the major histocompatibility complex on chromosome 6.
|Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli. |
Zhang, A J, et al.
J. Biol. Chem., 267: 1484-90 (1992) 1992
The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.
|Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. |
Cohen, P, et al.
Meth. Enzymol., 159: 427-37 (1988) 1988
|Phosphoprotein phosphatase inhibitor-2. Identification as a species of molecular weight 31,000 in rabbit muscle, liver, and other tissues. |
Roach, P, et al.
J. Biol. Chem., 260: 6314-7 (1985) 1985
Specific antibodies, raised to purified rabbit skeletal muscle inhibitor-2, were used to analyze for the presence of inhibitor-2 in extracts of rabbit skeletal, cardiac, and diaphragm muscles, liver, kidney, brain, and lung. Direct analyses of the extracts by "Western blotting" revealed several immunoreactive species, apparent molecular weights in the range 26,000-136,000, as well as species with the electrophoretic mobility of inhibitor-2, apparent molecular weight 31,000. When supernatants from boiled extracts were similarly analyzed, most of the immunoreactive material was lost and the species corresponding to inhibitor-2 became prominent. Liver and muscle were studied in more detail; immunoprecipitates from either boiled or unboiled extracts were analyzed by Western blotting. The dominant polypeptide now was the species of apparent molecular weight 31,000, corresponding to inhibitor-2. Higher molecular weight species (115,000 in muscle and 136,000 in liver) were also detectable. The amount of inhibitor-2 detected in immunoprecipitates was not greatly different whether unboiled or boiled tissue extracts were used. In addition, extraction of the precipitates by boiling released material that inhibited purified type 1 protein phosphatase. The results suggest that inhibitor-2 is widely distributed in rabbit tissues and is found predominantly as a form of apparent molecular weight 31,000. In particular, the study provides direct demonstration of a species in rabbit liver with similar properties to rabbit muscle inhibitor-2.
|The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities. |
Ingebritsen, T S and Cohen, P
Eur. J. Biochem., 132: 255-61 (1983) 1983
The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.