Metabolic fate of the major cell surface protein of normal human fibroblasts

The major cell surface protein of a strain of human fetal lung fibroblasts is a 220,000 dalton glycoprotein termed fibronectin. Fibronectin is released from fibroblasts into the culture medium with a half-time of about 25 hours. The release occurs with an initial (0–3 hours) rapid phase followed by a second (3–48 hours) slower phase. Release of fibronectin occurs in a stoichiometric fashion. All of the fibronectin released from the cells is quantitatively recovered from the culture media as a similar sized soluble glycoprotein. Thus, release of fibronectin from normal human fibroblasts results from mechanisms other than extensive degradation of the protein.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Dexamethasone selectively increases monoamine oxidase type a in human skin fibroblasts

The effects of several hormones known to affect monoamine oxidase activity $\text{in}\text{vivo}$ have been studied in living human skin fibroblasts grown in culture. Of the hormones tested, the synthetic glucocorticoid dexamethasone caused the greatest increases in activity at physiologic concentrations. Increases of 10–12 fold were observed after 8–9 days of exposure to 5 × 10^?8 M dexamethasone. This increase in activity was accompanied by a change in the relative proportion of the A and B types of activity in fibroblasts, from about 35% A:65% B in control cultures to 90% A:10% B in cultures exposed to dexamethasone. The increase in activity and the shift in the proportion of A and B activities could be accounted for almost exclusively by a specific increase in the number of Type A molecules.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

alpha7B integrin changes in mdx mouse muscles after L-arginine administration

Muscle fibers attach to laminin in the basal lamina using two mechanisms, i.e., dystrophin with its associated proteins and alpha7beta1 integrin. In humans, gene-mutation defects in one member of these complexes result in muscular dystrophies. This study revealed changes after L-arginine treatment of utrophin-associated proteins and the alpha7B integrin subunit in mdx mouse, a dystrophin-deficient animal model. In the two studied muscles (cardiac muscle and diaphragm), the alpha7B integrin subunit was increased in 5-week-old treated mice. Interestingly, the diaphragm histopathological appearance was significantly improved by L-arginine administration. These results highlight a possible way to compensate for dystrophin deficiency via alpha7beta1 integrin.     

Identification of rabbit microsomal cytochrome P-450 isozyme,form 1, as a hepatic progesterone 21-hydroxylase

Six highly purified forms of rabbit microsomal cytochrome P-450, isolated from hepatic microsomes, exhibit differences in the regiospecific metabolism of progesterone. Only one of the isozymes studied, form 1, catalyzes the formation of deoxycorticosterone from progesterone at an appreciable rate. This cytochrome P-450 isozyme may participate in the conversion of progesterone to deoxycorticosterone during pregnancy. All six forms of cytochrome P-450 catalyze 6β- and 16α-hydroxylation at the two concentrations of progesterone tested. Form 3b exhibits a lower apparent Km for 6β-hydroxylation than the other five.     

Induced changes in the affinity of 1,25-dihydroxyvitamin D3 receptors in chick intestine

The contents of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in plasma and intestinal mucose were increased by dietary calcium and by dietary phosphorus restriction. The concentration of intestinal occupied receptors for 1,25(OH)2D3 was higher in calcium-restricted birds. The affinity (association constant) of intestinal receptors for 1,25(OH)2D3 was lower in phosphorus-restricted chicks, as compared to control or calcium-restricted chicks. The number of binding sites were not influenced by dietary calcium or phosphorus restriction.     

Insulin stimulates phosphorylation of a heat-stable protein in rat adipose tissue

Insulin in rat adipose tissue acts to increase the phosphorylation about 2.5-fold of a low molecular weight protein in the cytosol designated phosphoprotein m. Isoproterenol had no effect on the phosphorylation of phosphoprotein m. Some of the properties of phosphoprotein m are: soluble in 1% trichloro acetic acid, heat-stable and has a molecular weight of 23,000 on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphoserine and phosphothreonine are the phosphorylated amino acid residues of phosphoprotein m. The physical and chemical properties of phosphoprotein m are similar to those of previously described inhibitor and modulator proteins.     

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

Susceptibility of platelet alpha-actinin to a Ca2+-activated neutral protease

Purified alpha-actinin from human platelets was digested with Ca2+-activated protease from muscle. The alpha subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, alpha-actinin is cleaved by the endogenous Ca2+-activated protease.     

Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.