A stereospecifically 18O-labelled deoxydinucleoside phosphate block for incorporation into an oligonucleotide

Fully protected diastereoisomers of deoxyguanylyl (3' leads to 5') deoxyadenosine stereospecifically labelled on phosphorus with oxygen-18 have been synthesized by oxidation of phosphite triester intermediates in the presence of 18O-labelled water. The diastereoisomers have been chromatographically separated and their absolute configuration at phosphorus determined. (Rp)-[18O]deoxyguanylyl (3' leads to 5')deoxyadenosine has been prepared by complete deprotection of the parent diastereoisomer of the Sp configuration. Methylation of the former compound permits assignment of the absolute configurations of the methyl esters of N1-methyldeoxyguanylyl (3' leads to 5') N1-methyldeoxyadenosine.     

Glycogen synthase kinase-2 and phosphorylase kinase are the same enzyme.

The primary structure of the nucleic acid from the branching enzyme 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase (2.5-S RNA) isolated from rabbit muscles has been elucidated. The polyribonucleotide consists of 31 nucleotides; the unique features of the polyribonucleotide are the unusually high content of modified nucleotides (32%) and guanine residues (40%). Apparently 2.5-S RNA belongs to a class of nucleic acids unknown up to now. It is the first time that the structure of a nucleic acid component from a ribonucleoenzyme has been defined. This work is a preprequisite for gaining insight into the intimate activating effect of the poly-ribonucleotide on the enzyme action.     

Site-specific antibodies block kinase A phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes.

Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP-dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino-terminal headpiece domains; in chicken smooth muscle desmin the most kinase A-reactive residues are ser-29 and ser-35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti-peptide antibodies directed against residues 26-36. The antibodies, which we call anti-D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26-36. Pre-incubation of desmin with affinity purified anti-D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75-80%. When antibody-treated IFs are subjected to phosphorylation, no filament break-down is observed after 3 hours. Thus anti-D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti-D26 at the mononucleated, myoblast stage. By 24 h post-injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation-induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.     

Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.     

Lack of phosphate incorporation into TN-I in live frog muscle

The phosphorylation of TN-I was investigated in muscles of live frogs injected with [³²P]orthophosphate. Isolation of TN-I was carried out by the rapid and specific technique of affinity chromatography developed by Syska, Perry, and Trayer [FEBS Lett.40, 253–257 (1974)] followed by gel electrophoresis in the presence of sodium dodecyl sulfate. No significant labeling of TN-I was found even in frogs which were exposed to the ³²P-treatment for several days. A comparison of the specific radioactivity of TN-I from resting and contracting frog muscle showed no change in the ³²P content of TN-I during muscle contraction.     

Inhibition of the phosphorylase kinase activity by ATP analogs and their binding to the enzyme subunits

The interaction between phosphorylase kinase (EC 2.7.1.38), isolated from rabbit skeletal muscles, and the ATP analogs with the modified triphosphate fragment: adenosine-5'-chloromethane pyrophosphonate (1), adenosine-5'-chloroethyl phosphate (2), adenosine-5'-bromethane pyrophosphonate (3), adenosine-5'-bromoethane phosphonate (4), adenosine-5'-chloroacetylaminomethane phosphonate (5), adenosine-5'-chloroacetylaminomethane pyrophosphonate (6) and adenosine-5'-chloromethane phosphonate (7), was studied. The compounds 1, 2 and 3 irreversibly inhibit the enzyme activity. In the presence of ATP the rate of inactivation is decreased. The radioactive compounds 1, 2 and 3 are stoicheometrically incorporated into the beta- and gamma-subunits of phosphorylase kinase. A correlation is shown to exist between the degree of the beta-subunit modification by compound 1 and the enzyme inactivation. The compounds 4, 5 and 6 inhibit the enzyme reversibly: in the presence of ATP complete protection of the enzyme activity is observed. The compound 7 does not affect the kinase activity; however, it binds itself to the beta-subunit of the enzyme. The binding of analogs 1 and 7 to the beta-subunit occurs at different sites. The data obtained are indicative of the catalytic role of the beta-subunit of phosphorylase kinase.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.     

Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn²⁺, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme.     

The incorporation of the phosphate esters of N-substituted aminoethanols into the phospholipids of brain and liver

1. The phosphate esters of dimethylaminoethanol and monomethylaminoethanol can be incorporated from their cytidine diphosphate esters into the phospholipids of brain and liver dispersions; the deoxycytidine nucleotides of the same bases are less effective precursors. 2. The cytidylyltransferases of brain and liver are less effective in forming the cytidine diphosphate esters of monomethylaminoethanol and dimethylaminoethanol than those of ethanolamine and choline.