Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals.Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation.Catalytic parameters such as time course, protein, ATP, Mg²⁺ concentration curves and K_m have been determined and were found to be similar in the reticulocyte and the erythrocyte.Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E₁ and prostaglandin E₂. Activation by prostaglandin E₁ was higher than that produced by prostaglandin E₂. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte.In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).     

Na+, K+-ATPase: evidence for the binding of ATP to the phosphoenzyme

Highly purified Na⁺, K⁺-ATPase of the dog kidney was reacted with Mg²⁺+³²Pi or Mg²⁺+³²Pi + ouabain. ³²P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K_0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na⁺. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

Decreased iodination of the red cell surface following phospholipase C treatment

Human red blood cells were treated with phospholipase C from Clostridium welchii. Lipase concentrations which produced <1% hemolysis and 10–15% hydrolysis of the membrane phospholipids reduced markedly ($\text{>80\%}$) the accessibility of mambrane proteins to the external surface as measured by lactoperoxidase-catalyzed iodianation.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Introduction of calcium chelators into isolated rat pancreatic acini inhibits amylase release in response to carbamylcholine

The Ca2+ chelators, EGTA and BAPTA, have been introduced into intact, isolated rat pancreatic acini using a hypotonic swelling method. This resulted in complete inhibition of amylase release, stimulated by carbamylcholine at a submaximal concentration and 82 - 85% inhibition at maximal concentrations. Acini swollen in the absence of Ca2+ chelators showed similar secretory responses to those of unswollen acini. Treatment of unswollen acini with chelators inhibited the maximum response to carbamylcholine by only 23%. The inhibitory effect of intracellular chelators was not due to ATP depletion or a lowering of the total cell Ca2+ content. Thus, these results provide the first direct demonstration that an increase in intracellular Ca2+ concentration is necessary for the stimulation of enzyme release from pancreatic acinar cells.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

Some kinetic properties of phosphorylated ATPase of sarcoplasmic reticulum formed in the absence of added alkali metal salts.

In the rat both hypothyroidism and diabetes decrease heparin-releasable liver lipase activity. This defect may be reversed by feeding a diet rich in polyunsaturated fatty acids. It is suggested that a diet-induced increase of membrane fluidity restores liver lipase activity, which contributes to the hypolipidemic effect of polyunsaturated fatty acids.     

Molecular diversity of calpastatin in mammalian organs

The crude homogenates of various human and porcine organs were subjected to immunoelectrophoretic blot analysis using affinity-purified anti-calpastatin antibody which specifically reacts with human erythrocyte 70 kDa calpastatin. Multiple immuno-reactive bands were revealed which ranged from 100 to 50 kDa. The results indicated the diversity of monomeric calpastatin molecules. The band patterns were different from one organ to the other. Among them, lung, heart and skeletal muscle were characterized by the predominance of 90-100 kDa calpastatin, having a common antigenicity to erythrocyte 70 kDa calpastatin. Such molecular diversity of calpastatins was also substantiated by enzymatic and chromatographic analyses.     

Rapid changes in chick liver acetyl-CoA carboxylase indicative of phosphorylation control

Liver fatty acid synthesis was suppressed 75,95 and 90% within 1, 2 and 4 hrs respectively of depriving chicks of food. Accompanying this rapid drop in lipogenesis was a marked reduction in acetyl-CoA carboxylase activity, i.e., 40 and 75% decrease after 2 and 4 hrs of fasting. Adding 10mM citrate to the crude liver supernatant, or incubating the supernatant at 37°, 30 min increased activity of the briefly fasted birds, but neither method restored carboxylase activity to fed level. Heat and citrate activation were additive and together resulted in an activity comparable to the fed condition. The heat-dependent activation was accelerated by exogenous phosphoprotein phosphatase, and completely blocked by 100 mM NaF. Thus, enhancement of carboxylase activity from liver of briefly fasted chicks appears to be a dephosphorylation process. This is the first report indicating acute changes in chick carboxylase activity may involve a phosphorylation-dephosphorylation mechanism.     

Phosphorylation of filamin and other proteins in cultured fibroblasts.

Incubation of subcellular fractions of fibroblasts with [³²P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.     

Requirement of trypsin inhibitor for measurement of 3-hydroxy-3-methylglutaryl coenzyme A reductase in intestinal mucosa of rat

A method was developed for the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the microsomal fraction of crypt cells and villi of rat intestinal mucosa. Addition of trypsin inhibitor to homogenizing and incubation media at a proper concentration appeared inevitable for measurement of the activity of the villi fraction. The reductase in crypt cells was also slightly enhanced by the addition of the inhibitor. Using this technique, the enzyme activity in villi was found to be as active as the crypt cell fraction. Since other types of protease inhibitors were not necessarily effective, it was suggested that specific enzyme(s) inactivates the mucosal reductase in the course of measurement.     

Involvement of hyposialylated immunoglobulins in the inactivation of alpha 1-proteinase inhibitor

The elastase inhibitory capacity of alpha 1-proteinase inhibitor (alpha 1-PI) was measured, using a direct and reproducible method, with phagocytic cells maintained in the tissue culture plate through the assay. The oxidative inactivation of alpha 1-PI is known to be mediated by the action of myeloperoxidase (MPO). The fact that hyposialylated IgG (hs IgG) induce the release of MPO prompted us to investigate the effects of such hs IgG on the inhibitory capacity of alpha 1-PI. The results show that 1-PI inactivation was observed only when phagocytic cells were activated by aggregated hs IgG, and not by unaggregated hs IgG. These observations indicate that hyposialylation should be completed by aggregation to perpetuate the oxidative reactions characteristic of inflammatory diseases.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

Quantitation and characterization of the microtubule associated MAP2 in porcine tissues and its isolation from porcine (PK15) and human (HeLa) cell lines

A radioimmunoassay developed for the microtubule associated protein MAP₂ shows that this protein, or related polypeptides are present in all the porcine tissues studied. Nervous tissues (brain, 11 μg MAP₂/mg protein; cerebellum, 9.7 μg MAP₂/mg protein) contain much higher levels of MAP₂ than non-nervous tissues (kidney, 104 ng MAP₂/mg protein; lung 89 ng MAP₂/mg protein; spleen 66 ng MAP₂/mg protein; thyroid 21 ng MAP₂/mg protein; liver 9.7 ng MAP₂/mg protein). A heat resistant protein doublet of 300,000 with the ability to promote microtubule polymerization has been purified from pig kidney cells by affinity chromatography using MAP₂ antibodies. Using a similar purification method a protein of 200,000 daltons has been isolated from Hela cells.     

Stimulation of erythroid cells adenylate cyclase by soluble factors

Soluble factors obtained from human, rat and rabbit erythroid cell lysates are capable to stimulate basal and hormone activated adenylate cyclase of erythroid cell membranes from homologous sources. Extensive dialysis and removal of hemoglobin from the soluble factors do not modify their activity. Human erythrocyte soluble factors stimulate the human reticulocyte enzyme. Nevertheless human erythrocyte adenylate cyclase is not stimulated by either of the soluble factors. The presence of active soluble factors in human erythrocytes where the adenylate cyclase is no longer sensitive to these factors, as well as to guanylnucleotides or protaglandins, indicates that the enzyme has been altered during the maturation processes.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Protein phosphorylation in pancreatic islets: evidence for separate Ca2+ and cAMP-enhanced phosphorylation of two 57,000 Mr proteins

There is a phosphopeptide that has an Mr of 53,000 to 60,000 in insulin-secreting tissues and there is general agreement that this peptide can be phosphorylated in a calcium-dependent manner. The present report shows that there are at least two phosphoproteins with Mr's near 57,000 in rat pancreatic islet cytosol. One peptide has an Mr of 57,000, a pl of 7.5 - 8 and is phosphorylated in a Ca2+-enhanced manner, and the other has an Mr of 54,000, a pl of 5 - 5.5 and is phosphorylated in a cAMP-enhanced manner, as judged by two-dimensional polyacrylamide gel electrophoresis. Sepharose 4B chromatography indicated that the former polypeptide resides in a native protein complex that has an Mr of about 500,000 and the latter in a complex that has an Mr of about 180,000. Tritiated azido cyclic AMP binds to an islet polypeptide that has an Mr of 54,000. The results suggest that Ca2+ and cAMP could regulate stimulus-secretion coupling in pancreatic islets via protein phosphorylation.