The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

A monolayer study of the reaction of trinitrobenzene sulphonic acid with amino phospholipids.

The reaction of trinitrobenzene sulphonic acid with amino phospholipids, and in particular phosphatidylethanolamine has been studied by the monolayer technique. Injection of trinitrobenzene sulphonic acid under a monolayer of amino phospholipid results in an increase in surface pressure. The rate and extent of the pressure change is greatly affected by the initial surface pressure, the fatty acid composition of the lipid, and the presence of other non-reactive lipids, especially negatively charged phospholipids. The extent of the reaction was measured with 32P-labelled phospholipids isolated from Bacillus subtilis. Only about 80% of the phosphatidylethanolamine in the monolayer could be converted to its trinitrophenyl derivative. In the presence of negatively charged phospholipids such as cardiolipin or phosphatidylglycerol, a further 20% decrease in the trinitrophenylation of phosphatidylethanolamine was found. The pressure increase occurring during trinitrophenylation could also be correlated with the extent of the reaction by comparison of the force-area curves of pure phosphatidylethanolamine, its trinitrophenyl derivative and mixtures of both compounds. The data may offer an explanation for the observation that incomplete labelling of amino phospholipids frequently occurs in natural membranes and furthermore indicate that the use of chemical labelling techniques in the study of lipid asymmetry in biological membranes must be approached with great caution.     

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.     

A new lethal syndrome with cloudy corneae,diaphragmatic defects and distal limb deformities

Summary Two female sibs are reported with a possibly new lethal malformation pattern, the major anomalies of which are: coarse face with small eyes and cloudy corneae, cleft soft palate, hypoplasia and absence of lobulation of both lungs, diaphragmatic defects, digitalisation of thumbs and distal limb deformities.     

A chromosomal abnormality (21q-) in primary thrombocytosis

Summary A patient with primary thrombocytosis was found to present an acquired deletion of the long arm of chromosome 21 (21q-). A similar observation reported in the literature is hereby confirmed.     

Primary mesodermal dysgenesis of the cornea (Peters' anomaly) in two brothers

Summary In this report two brothers with a severe form of Peters' anomaly, i.e., primary dysgenesis mesodermalis of the cornea, are described. The thirddegree consanguinity of the parents is an additional argument for the autosomal recessive inheritance of this condition.     

Qualitative and quantitative study of the growth and cell surface properties of Huntington's disease fibroblasts and age-matched controls

Summary Different growth-and cell surface properties of cells grown from skin biopsy samples from 25 Huntington's chorea patients, 22 at risk patients, and agematched controls were examined in a blind study. The number of explants obtained from each biopsy was carefully controlled. The nature (epithelial or fibroblastic) of the initial outgrowth was scored on all explants and the mean number of cells obtained from each explant after 3 weeks in culture was determined after trypsinization. During the next 15 passages, various parameters characterizing cell growth were studied. Maximal cell densities and growth rates were examined using a standard plating density of cells at each passage. These growth properties were also examined in three media: DME-NCS, DME-FCS, and Ham's F10 fetal calf serum (FCS). In addition, the effects of high temperature (40°C) and the addition of insulin to the medium on the growth and protein content of the cells was examined. These results, containing 152 measurements on 81 cases, were examined using a phased multivariate analysis. Blind cluster analysis showed that the clusters were nonrandomly determined. Factor analysis indicated the initial growth parameters to be among the most discriminating. Discriminant analysis, however, did not prove to be of any diagnostic use, since the external factors masked any possible genetic difference. Two possible sources of bias were identified: the size of the biopsy and the type of culture medium. The cell surface properties of HD and control fibroblasts were further investigated using two different techniques. The adhesion of single HD cells to either HD cell layers or normal cell layers revealed no significant differences. Intradermal immunization of rabbits with whole fibroblasts resulted, with both cell lines, in a polyspecific antiserum containing antibodies directed against a number of surface components. When HD and control fibroblasts were compared with the two antisera, no qualitative or quantitative differences in the precipitation patterns obtained using crossed immunoelectrophoresis, (CIE) were found. Our investigations did not allow us to discriminate HD cultures from controls by any of the qualitative and quantitative parameters tested.     

Lysophospholipase and lysophosphatidylcholine:Lysophosphatidylcholine transacylase from rat lung: Evidence for a single enzyme and some aspects of its specificity

An enzyme preparation was isolated from rat lung cytosol with the capability to transfer the fatty acyl chain from 1-acyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-acyl-sn-glycero-3-phosphocholine. The evidence presented to indicate that a single protein confers both activities includes: (a) both normal and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis showed a single protein band, and (b) heat treatment and preincubation with increasing amounts of diisopropylfluorophosphate resulted in concomitant loss of fatty acid and phosphatidylcholine formation. The enzyme converted 1-[9,10-³H₂]stearoyl-sn-glycero-3-phospho[¹⁴C-methyl]choline into phosphatidylcholine with an isotopic ³H/¹⁴C ratio twice that of the substrate, even when an excess of unlabeled fatty acid was present. The acyl group from palmitoyl-propanediol (1,3)-phosphocholine and palmitoyl-propanediol (1,3)-phosphoethanolamine could be transferred to lysophosphatidylcholine acceptor to yield phosphatidylcholine. Neither acylglycerols and cholesterol nor glycero-3-phosphate and glycero-3-phosphocholine served as acyl acceptors. Lysophosphatidylethanolamine and lysophosphatidyglycerol were converted also into the corresponding diacylphospholipids. Palmitoyllysophosphatidylcholine is preferentially converted into phosphatidylcholine when compared with stearoyllysophosphatidylcholine. The possible involvement of the enzyme in the synthesis of dipalmitoylphosphatidylcholine for the production of lung surfactant is discussed.