Beta-cell dysfunction in 48-hour glucose-infused rats is not a consequence of elevated plasma lipid or islet triglyceride levels.

The abnormal insulin secretion found in human diabetics and animal models of diabetes has been attributed to the deleterious effects of chronic hyperglycemia and/or elevated circulating levels of nonesterified fatty acids (NEFAs). In this study, abnormal glucose-induced insulin secretion (GIIS) was generated by a 48-hour infusion of glucose and assessed by the isolated perfused pancreas technique. In these hyperglycemic animals, abnormal GIIS is accompanied by a decrease in plasma NEFAs, while plasma and, more importantly, islet triglycerides remain at levels comparable to those in the controls. It is concluded that the abnormal insulin secretion in this glucose infusion model was likely caused by 48 hours of hyperglycemia and not by changes in circulating or islet lipids.

Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems.

We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein. Expression of the human enzyme in Sf-9 insect cells after infection with recombinant baculovirus encoding the human squalene synthase gene resulted in detection of substantial enzymatic activity in cell lysate preparations. Following extraction from the Sf-9 cells, the human enzyme was purified to near homogeneity utilizing a series of ion-exchange chromatography steps with an overall yield of purified protein of approximately 5 mg per liter of Sf-9 cell culture. The purified enzyme was characterized through steady-state kinetic and physical measurements and the kinetic constants are consistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnesyl pyrophosphate and has a Kis value of 250 pM (@ [NADPH] = 5 mM). Inhibition experiments with zaragozic acid C at low (approximately 0.5 x Km) and high (approximately 10 x Km) concentrations of NADPH indicated that the inhibitor does not bind in the enzyme's NADPH binding domain. These studies demonstrate that the human enzyme can be prepared from baculovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic, and structural characterization.

Cloning, expression, and characterization of human apolipoprotein(a) kringle IV37.

A portion of kringle IV37 (KIV37) of apolipoprotein (a), (apo(a)), was polymerase chain reaction-cloned from human liver cDNA. The protein product of this clone was expressed in Escherichia coli as a poly histidine fusion protein. Based on recovery of purified fusion apo(a) KIV37 protein expression levels were estimated to be 10 mg/g of E. coli cell paste. Mass spectral analysis showed the molecular mass of fusion apo(a) KIV37 to be 12,260 +/- 1 daltons. Almost all fusion apo(a) KIV37 was expressed as inclusion bodies and had to be refolded. Fusion apo(a) KIV37 was isolated from the inclusion bodies and purified by lysine-Sepharose affinity chromatography by eluting with 0.2 M epsilon-aminocaproic acid. The fusion protein was treated with thrombin to yield a homogeneous, functional apo(a) KIV37 domain composed of 92 amino acids having a molecular mass of 10,510 +/- 1 daltons. N-terminal protein sequencing and amino acid analysis have confirmed the sequence and composition of apo(a) KIV37. The molar extinction coefficient, epsilon, for apo(a) KIV37 was determined to be 3.1 x 10(4) M-1 cm-1, and the pI was measured to be 6.7 +/- 0.1. In addition, the dissociation constants, Kd, for a series of 11 lysine analogs have been determined by measuring the change in intrinsic fluorescence of apo(a) KIV37 upon saturable binding with these compounds. Kd values ranged from 4.2 +/- 0.9 microM for trans-4-(aminomethyl)cyclohexanecarboxylic acid to 4.6 +/- 0.4 mM for L-arginine. Apo(a) KIV37 binds to plasmin-treated fibrinogen with an EC50 value of 14 +/- 1.2 microM and prevents the binding of Lp(a) to plasmin-treated fibrinogen with an IC50 value of 16 +/- 6 microM. Lp(a) binds to the plasmin-treated fibrinogen surface with an EC50 value of approximately 1.0 +/- 0.3 nM. These studies demonstrate that apo(a) KIV37 can be expressed at high levels, refolded properly, and used as a fully functional lysine-binding domain. In addition, these results also demonstrate that apo(a) KIV37 provides the major interaction of Lp(a) with fibrinogen. One additional weak binding site in Lp(a) is adequate to describe overall Lp(a) binding to fibrinogen.