The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.     

Internally elongated rodent α-crystallin A chain: Resulting from incomplete RNA splicing?

αA^Ins, an elongated α-crystallin A chain previously observed in rat, was present beside the normal αA chain in mouse, gerbil and hamster, which places its origin at least 30 million years ago. Like in rat the sequences of golden hamster αA^Ins and αA were found to be identical, apart from the internal insertion of 22 residues in αA^Ins. The hamster chains only differed from the rat chains by a single substitution in the inserted sequence of αA^Ins. The origin of αA^Ins, by insertion of 22 residues in an otherwise unchanged αA chain, and its rigid evolutionary conservation are most easily explained by assuming the incomplete removal of a putative intervening sequence from the precursor mRNA of αA, leaving an intracistronic insert of 66 nucleotides in part of the eventually translated mRNA.     

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.     

Activation of Phycomyces adenosine 3', 5'-monophosphate phosphodiesterase by blue light.

Cyclic AMP phosphodiesterase can be extracted from sporangiophores of $\text{Phycomyces}\text{blakesleeanus}$. Activity is enhanced by 1–10 μM FAD and FMN but not by riboflavin. Moderate intensity of blue light also activates the enzyme, especially in the presence of 1mM GTP. The enzyme must be extracted and stored in the absence of blue light for this result. Forty times the intensity of red light has no effect. This finding is consistent with the very sudden transient drop in cyclic AMP level upon light stimulation in the intact sporangiophore.     

Antimetabolic activities of 2-fluoro-L-histidine.

2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by ³H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.     

The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle.

Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorylated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419--426]. Inhibitor-1 was purified by a heat treatment at 90 degrees C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally rechromatography of the phosphorylated protein on DEAE-cellulose, The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seven days corresponding to an overall yield of 15-20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 micron, which is at least as high as the concentration of phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibrium centrifugation was 19200 and by amino acid analysis was 20800. These values were lower than the mol. wt 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weight of 60000 estimated by gel filtration on Sephadex G-100. The gel filtration behaviour, stability to heating at 100 degrees C and amino acid composition suggest that inhibitor-1 may possess little ordered structure. The phosphorylated from of inhibitor-1 contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with the previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Thr- [Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEBS Lett. 76, 182-186].the phosphorylated form of inhibitor-1 inhibited phosphorylase phosphatase activity (0.02U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000-2000 times less effective as an inhibitor.     

Biochemical characterization of density-separated human erythrocytes.

A simple, reproducible method for the separation of human erythrocytes, described recently (Murphy, J. R. (1973) J. Lab. Clin. Med. 82, 334-341) has been utilized for the purpose of obtaining a wide range of biochemical data on these cells. Using phthalate ester density centrifugation of the fractions obtained by Murphy's method, we established that the cells were separated exclusively on the basis of their densities. Data on a wide range of biochemical and hematological parameters, when compared with previously reported density separation procedures showed that this simple technique can be used to fractionate the cells according to their densities (age) in their own plasma. Cells of increasing density consistently and reproducibly exhibited an increase in hemoglobin concentration, a moderate elevation in Na+ and a decrease in the following: K+, acetylcholinesterase, sialic acid, membrane protein, 2,3-diphosphoglycerate, ATP, cholesterol, phospholipid, mean corpuscular volume and critical hemolytic volume, However, no change in mean corpuscular hemoglobin was evident. The observed differences were not artifacts of the centrifugation process. This was determined in recentrifuged top fractions from which new top and bottom cells were obtained. The latter cells resembled the top fraction from which they were obtained, rather than the original bottom fraction. Whereas the parameters mentioned above exhibited consistency and reproducibility, such was not the case with the ATPase values. Depending on the cell density group examined and/or buffer as well as other conditions, significant variability in the activity levels of the ouabain sensitive, as well as the Ca2+ -stimulated ATPase, was observed. Use of these enzyme activities as indicators of cell age must be viewed with caution.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with ¹²⁵I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.