Michihiro Sumida

Biomodulator-mediated susceptibility of endogenous lipid droplets from rat adipocytes to hormone-sensitive lipase.

The amount of fatty acid release by a fat cell homogenate without pretreatment with epinephrine was found to be slightly more than that released from fat cells by epinephrine, suggesting that fat cells contain high lipolytic activity even in the absence of lipolytic agents. Fat cells contain high hormone-sensitive lipase activity (1383 mumole free fatty acids/g/hr) in the absence of epinephrine, and addition of epinephrine to the cells did not increase the activity, significantly. Like epinephrine, DBcAMP and/or theophylline also elicited marked release of glycerol from fat cells without activating the hormone-sensitive lipase activity. However, although fat cells contain a large amount of hormone-sensitive lipase, lipolysis was negligible in the absence of these lipolytic agents. These results suggest that lipolytic agents such as epinephrine, DBcAMP, and theophylline induce lipolysis in fat cells through some mechanism other than activation of hormone-sensitive lipase and that in the absence of lipolytic agents, some system in fat cells inhibits lipolysis of endogenous lipid droplets by hormone-sensitive lipase. The lipid droplets in fat cells consist mainly of triglyceride with phospholipids, cholesterol, carbohydrate, and protein as minor constituents. The phospholipid fraction was found to consist of 75% phosphatidylcholine and 25% phosphatidylethanolamine. Of the minor constituents of endogenous lipid droplets, only phosphatidylcholine strongly inhibited hormone-sensitive lipase activity in a [3H]triolein emulsion. These results suggest that phosphatidylcholine in endogenous lipid droplets may be responsible for inhibition of hormone-sensitive lipase. Then, a cell-free system was established in which epinephrine, DBcAMP, and theophylline stimulated lipolysis of endogenous lipid droplets from fat cells by lipase solution. In this system, these lipolytic agents did not induce lipolysis in the absence of added lipase. Lipolysis in the mixture of the endogenous lipid droplets and lipase solution was accelerated by phospholipase C with concomitant loss of epinephrine-induced lipolysis. After pretreatment of the endogenous lipid droplets with phospholipase C, these lipolytic agents no longer induced lipolysis. Pretreatment of the endogenous lipid droplets with phospholipase C reduced their phospholipid content with the formation of phosphorylcholine, but did not affect their triglyceride and cholesterol contents. Treatment of the endogenous lipid droplets with phospholipase D did not affect lipolysis in the cell-free system. These results suggest that phosphatidylcholine in the endogenous lipid droplets may inhibit their lipolysis by hormone-sensitive lipase in fat cells and also be involved in the mechanisms of the stimulatory effects of epinephrine, DBcAMP, and theophylline on lipolysis.

Gene Expression and Fat Deposit in Primary Cultures of Rat Meibomian Gland Cells

Meibomian glands synthesize and store triglyceride, wax- and cholesteryl-ester, and secrete them onto the ocular surface as tear stabilizers. Since meibomian gland dysfunction causes dry eye and other ocular surface diseases,1 it is crucial to clarify molecular mechanisms of lipid metabolism and gene expression of specific lipid metabolizing proteins in these glands. Although a number of enzymes involved in lipid metabolism are presumed to be produced by the tissue, few of them have as yet been identified. In the structurally similar sebaceous gland, peroxisomal proliferator-activated receptor (ppar)-gamma, a lipid-related transcription factor, was found,2 and reaction mechanisms of fat metabolism similar to those in adipose tissues were indicated.

SITE-DIRECTED MUTAGENESIS AND STEADY-STATE KINETIC ANALYSIS OF MUTANT ENZYMES OF HUMAN ADENYLATE KINASE

Site‐directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C‐terminal segment (Val182, V186, and Leu193). Five mutants (H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193‐194) were generated and analyzed by steady‐state kinetics. H36T, K55G, and L193Stop mutants showed an increase of Km values (19.8‐, 19.7‐, and 11.3‐fold) for AMP2‐ compared to that for the wild‐type enzyme, and these residues appeared to interact with AMP2‐. V182G showed an increased Km value (7.4‐fold) for MgATP2‐. Therefore, V182 may be essential for interaction with MgATP2‐. V186S increased the Km value (7.0‐ and 7.5‐fold) for MgATP2‐ and AMP2‐. V186 may thus interact with both substrates. The C‐terminal domain of AK appears to be essential for MgATP2‐ and AMP2‐ binding.

Catalytic roles of lysines (K9, K27, K31) in the N‐terminal domain in human adenylate kinase by random site‐directed mutagenesis

To elucidate lysine residues in the N‐terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site‐directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady‐state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2‐. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2‐. In human AK, the ε‐amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2‐ and AMP2‐ during transition state.

PRE-STEADY STATE KINETICS OF E∼P FORMATION AND DECOMPOSITION BY Ca2+, Mg2+-ATPase IN BOVINE AORTA MICROSOMES

Publisher Summary It has been observed that vascular smooth muscle contains intracellular organelles, including sarcoplasmic reticulum(SR), which could regulate the cytosolic Ca2+ concentration that activates the contractile apparatus. Microsomal fractions isolated from the vascular smooth muscle have been shown to take up Ca2+ ions coupled to ATP hydrolysis; thus, these fractions have been suggested to be the counterparts of SR found in both skeletal and cardiac muscles. The chapter discusses a study to characterize the rates of the E∼P formation and decomposition using the rapid acid quench technique and to demonstrate presteady state profiles of phosphorylation identical to those found in skeletal and cardiac SR.