Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Effect of depletion of bicarbonate or phosphate ions on insulin action in rat adipocytes. Further characterization of the receptor-effector system

In the preceding paper (Shechter, Y., and Ron, A. (1986) J. Biol. Chem. 261, 14945-14950) we have shown that in fat cells, prepared and maintained in an isotonic buffer (pH 7.4) containing neither phosphate nor bicarbonate anions (Buffer A), the dose-response curve to insulin shifted to the right by about 2 logarithms and insulin binding affinity or capacity was only slightly decreased. In the current paper we demonstrate that progressive loss of insulin binding, either by treatment with trypsin or preincubating the cells with isoproterenol, correlates well with the reduced ability of the cells to elicit maximal lipogenesis in response to insulin. We further demonstrate in the "new" system that: the dissociation of labeled insulin from fat cells is not accelerated by the inclusion of unlabeled insulin in the medium; termination of lipogenesis in Buffer A occurs immediately; ligand-induced receptor internalization is grossly defective; and insulin is unable to stimulate lipogenesis at 15 degrees C. The data support the hypothesis that in the new experimental system all measurable binding sites are linked to a coupling mechanism. Each site behaves as an independent, separate entity and there are no site to site interactions. This leads to a linear relationship between binding and bioactivation, lack of negative or positive cooperatively, accelerated rate of termination, defective internalization, a shift to the right in the dose-response curve to insulin, and a lack of insulin response at a lower temperature. In more general terms, the study indicates that all measurable insulin receptors are chemically homogeneous in their potential capability to be coupled to an insulin effector (biologically relevant) system, and they do so under particular experimental conditions.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

Follicle-stimulating hormone enhances somatomedin C binding to cultured rat granulosa cells. Evidence for cAMP dependence

The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard.     

Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues

Two proteins of Mr = 58,000 and 59,000, respectively, were purified from 4 M guanidinium chloride extracts of articular cartilage by dissociative CsCl-density gradient centrifugation followed by gel chromatography on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The two proteins differ in ionic properties and only the one with Mr = 59,000 bound to the ion exchanger. Although the two proteins showed dissimilar peptide patterns after proteolysis, their amino acid composition was similar, with very high contents of leucine and aspartic acid/asparagine. The two proteins showed no cross-reactivity in radioimmunoassays. By use of these assays, the proteins were demonstrated in extracts of most connective tissues, with high contents of about 0.1% of tissue wet weight determined in several types of cartilage. Among the non-cartilage connective tissues, tendon and sclera had the highest contents of the proteins, i.e. about 0.1% of the tissue wet weight. Bone extracts, on the other hand, contained insignificant amounts of the proteins. Only the Mr = 59,000 protein was detected in serum, its concentration being about 33 micrograms/l. Both proteins were shown to be localized in the extracellular matrix of cartilage, predominantly in the territorial matrix, by using indirect immunofluorescence.     

Reflex cardiovascular responses caused by stimulation of pulmonary C-fibers with capsaicin in dogs

The purpose of these studies was to determine quantitatively the reflex cardiovascular responses to stimulation of the pulmonary C-fibers in dogs. We used a preparation in which the airway, pulmonary artery, and the pulmonary veins to the left lung were cannulated in situ. Ventilation and perfusion of the right lung maintained the animal in relatively normal homeostasis. Capsaicin, a decylenic acid amide of vanillylamine that selectively stimulates nerve endings of unmyelinated fibers (C-fibers), was injected into the left pulmonary artery in 5-ml boluses. Maximal reflex responses were obtained with concentrations as low as 0.8-1.6 X micrograms-1 X kg-1. Heart rate, hindlimb resistance, and left ventricular contractility were lowered transiently (the maximal responses showing declines of 40, 13, and 15.2%, respectively). As a result of these changes, combined with vasodilation in other resistance vessels, cardiac output fell 28% and blood pressure fell 35%. Interrupting the afferent neural pathway by severing the ipsilateral cervical vagus nerve eliminated these responses, confirming the distribution of their reflex origin. Although the role of these reflexes in homeostasis has not been decided, the results of this study suggest that the lungs of dogs, if appropriately stimulated, potentially can exert a major inhibitory influence on the neural regulation of cardiovascular function.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin

An Mr 19 000 protein (destrin) that has the ability to rapidly depolymerize F-actin in a stoichiometric manner was purified from porcine kidney by sequential chromatography on DNase I-agarose, hydroxyapatite, and Sephadex G-75. Its actin-depolymerizing activity is reversibly controlled by changes in KCl concentration but is insensitive to Ca2+ concentration. The rate of depolymerization of F-actin by destrin is much faster than that of spontaneous depolymerization induced by dilution and is not markedly decreased by the addition of end-blocking reagents such as cytochalasin B. These results suggest that destrin depolymerizes F-actin by interacting directly with F-actin protomers. Binding of muscle tropomyosin to F-actin slows down the rate of destrin-induced depolymerization of F-actin by about 30-fold. The data suggest that the destrin-induced depolymerization occurs from the ends of F-actin when F-actin is complexed with tropomyosin, but it takes place from the entire length of F-actin in the absence of tropomyosin.