Studies of corticotropin receptors on rat adipocytes

Synthetic [¹²⁵I]-Tyr²³, Phe², Nle⁴-adrenocorticotropin (ACTH)-(1–38) ([¹²⁵I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 ± 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [¹²⁵I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent K_D = 170 ± 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent K_D (143 ± 16.5 pm). The number of receptors per adipocyte was quite low (521–841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent K_m being 145–177 pm. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent K_D = 446 ± 77 pM) and the binding capacity was also significantly enhanced (1663 ± 208/cell) without any change in the apparent K_m for glycerol release (187 ± 7.1 pm).     

Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4. The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively. Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells. Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus. Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity.     

Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase indicates the requirement for the basic form of an ionizable group in the enzyme-substrate complex with a pK of 6.6. This pK is unaltered from 10 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, it reduces the concentration of Mn2+ (1 mM) and isocitrate (20 mM) required to produce the same decrease in the inactivation constant. These observations imply that the modification occurs at the substrate-binding site. Experiments employing [1-14C]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per subunit (40 000), concomitant with the complete inactivation of the enzyme. The radioactive modified enzyme, after removal of excess reagent by dialysis, was exhaustively digested with proteolytic enzymes. High voltage electrophoretic analyses of the hydrolysate at pH 6.4 and 3.5 yield two major radioactive spots with approximately equal intensity, which correspond to gamma-glutamylglycine and beta-aspartylglycine, the ultimate products of reaction with glutamic and aspartic acids, respectively. Modification in the presence of manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.     

Sub-mitochondrial location of Ruthenium Red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca²⁺ transport in the absence and in the presence of P_i as well as in the presence of N-ethylmaleimide to prevent P_i cycling, succinate-supported respiration in the absence and in the presence of ADP, the ΔE and −59 ΔpH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, α-glycerophosphate dehydrogenase, P_i content and the effect on the `resting' rate of respiration of repeated additions of a fixed Ca²⁺ concentration. 2. Ca²⁺ transport either in the presence or in the absence of added P_i and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for ΔE and −59 ΔpH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca²⁺ transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca²⁺, as measured by the effect of repeated additions of Ca²⁺ on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of α-glycerophosphate dehydrogenase was similar to that of Ca²⁺ transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem. 25, 379–396], it is concluded that the Ruthenium Red-sensitive Ca²⁺- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca²⁺ transport and the biogenesis of mitochondria is discussed.     

Studies on catalytic properties of purified high molecular weight pancreatic lipase.

The properties of the 500-fold purified high-molecular-weight lipase have been studied. The rate of hydrolysis of the triglycerides decreases with increasing fatty acid chain length. The lipolytic activity also increases with increase in unsaturation in the fatty acyl moiety. Diglycerides are hydrolyzed at more than twice the rate for triglycerides while monoglycerides are not hydrolyzed. Methyl esters are generally hydrolyzed at a higher rate which increases with increasing chain length of the fatty acid but the enzyme does not act on phospholipids. Emulsifying agents such as Tween 20, gum arabic, and albumin increase the rate of hydrolysis. Metal ions such as Hg2+, Zn2+, Cu2+, and Fe2+ strongly inhibit the lipolytic activity of the high-molecular-weight lipase while Ca2+ or Mg2+ by themselves show no stimulating effect. Treatment of the high-molecular-weight lipase with P-chloromercurybenzoate inhibits hydrolytic activity by 70% while iodoacetic acid has no effect.     

Evidence for the stimulation of dolichol and mannolipid synthesis by glucocorticoids in HeLa cells.

The effects of addition of 1 microM-dexamethasone on the rate of transfer of mannose from GDP-mannose into mannolipid have been studied in HeLa cell cultures. Concurrent with an increase in incorporation of mannose into glycoproteins, the incorporation of mannose from GDP-mannose in vitro into mannolipid and dolichol-linked oligosaccharides was increased after dexamethasone treatment. Stimulation of mannolipid synthesis showed a correlation with the 11 beta, 17 alpha, 21-trihydroxy structure of C21 steroids. Dexamethasone treatment also resulted in an increased incorporation of acetate into dolichol and dolichyl phosphate. The results suggest that the effect of dexamethasone on the cell-surface glycoprotein accumulation is related to increased sugar-linked dolichol synthesis.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. II. Urease in a packed-column differential reactor system.

Urease from Jack bean was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column differential reactor. To facilitate comparison, the urease was immobilized by both diazo and glutaraldehyde coupling. The kinetic properties of immobilized urease were similar to those of the soluble enzyme and different immobilization methods did not appreciably alter the kinetic properties. The affects of three different amino acid activators appear to follow predictions obtained from a relatively simple competitive model, except at very low substrate levels.     

Divergent effects of adrenocorticotropin and melanotropin on isolated rat and rabbit adipocytes.

The stimulation of lipolysis in isolated rat and rabbit fat cells by adrenocorticotropin (ACTH) and alpha-melanotropin has been studied. The concentration of alpha-melanotropin required for half maximal stimulation is 0.23 times that of ACTH in rabbit adipocytes but as high as 1140 times that of ACTH in rat fat cells. Chemical modification of the tryptophan residue in ACTH and melanotropin resulted in a loss of lipolytic activity in rat adipocytes and an increase in lipolytic potency in rabbit fat cells. These differences between rat and rabbit fat cells were evident when stimulation of cyclic AMP synthesis was measured in isolated cells or ghosts. The results are discussed in terms of the difference in the hormone receptors of the fat cells of the two species.