The kinetic properties of Mg2+-ATPase of bovine brain kinesin in the steady-state were studied in the absence of microtubules. A double-reciprocal plot of kinesin Mg2+-ATPase activity vs. ATP concentration indicated two distinct Km values, 128 and 3.3μM. ATPase activity decreased with increase in pH from 6.0 to 7.0, increased as the pH was increased to 8.0, and then gradually decreased above pH 8.0. The rate of ATP hydrolysis was also affected by the KCl concentration. ATPase activity decreased with an increase in KCl concentration from 15 to 50mM, increased as the KCl concentration was increased further, and then decreased when KCl exceeded 200mM. From an Arrhenius plot of temperature dependency of Mg2+-ATPase activity, activation energy was estimated to be 25.3J/mol between 45 and 23°C and 87.9J/mol between 23 and 6°C. Both heavy and light chains in kinesin were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a 2.8-fold activation of Mg2+-ATPase in the absence of microtubules. These properties are more like smooth muscle myosin than skeletal muscle myosin.
A serine protease was earlier purified to homogeneity from a culture extract of mites and its possible involvement in the allergic reaction was proposed [K. Takahashi et al. (1990): Int. Arch. Allergy Appl. Immun., 91, 80-85]. The serine protease thus obtained was found in the present study to be trypsin-like protease and had high substrate specificity towards the synthetic substrate for blood coagulation factor XIIa. The enzyme gave rise to rhythmic contraction of rat uterine horns in factor XII-deficient human plasma, but not in prekallikrein-deficient plasma. These findings lead us to conclude that this enzyme activates the kallikrein-kinin system in plasma through hydrolysis of prekallikrein in the plasma.
We previously constructed novel liposomes for efficient transfection of an entrapped gene into mammalian cells [Koshizaka, T., Hayashi, Y., and Yagi, K. (1990) J. Clin. Biochem. Nutr., 7, 185-192]. By encapsulation of histone, a nuclear protein, into the above liposomes at a histone/DNA ratio (w/w) of 0.2, the expression in COS-1 cells of β-galactosidase activity by the plasmid pCH110, which contains the lacZ gene under the control of the SV40 early promoter, was found to be increased by -50%. The increased expression efficiency seems to be due to the efficient transfer of the plasmid to the nucleus after its incorporation into the cells, probably through the protection of the DNA by its binding with histone and/or accelerated transfer of the DNA to the nucleus by this protien.
The effect of feeding of cholesterol equivalent to 7.692 and 10.026mg/kg/day on plasma lecithin: cholesterol aryl transferase activity in rats was examined. The enzyme activity was increased significantly in the cholesterol-fed rats (p<0.01) when compared with the level in the control animals fed a normal diet and was found to be dose (amount of cholesterol incorporated) dependent. The former group also showed relative significant increases in the plasma levels of cholesteryl ester and lysolecithin (p<0.05) and decreased levels of lecithin (p<0.01). Changes in free cholesterol levels were, however, not statistically significant (p>0.05) in the cholesterol-fed rats. The implications of these findings are discussed with respect to atherogenesis.
Our previous report suggested that increased serum angiotensin-converting enzyme (ACE) activity plays a central part in the pathogenesis of essential hypertension in man. In the present study, we investigated whether such findings can be seen in spontaneously hypertensive rats (SHR). The results suggested that serum carboxypeptidase N and kallikrein, rather than ACE, plays a central part in this experimental model. The ACE activity was decreased rather than increased in spontaneously hypertensive rat when compared with that in Wistar-Kyoto rats.
The effect of 4-aminopyrazolo [3, 4-d]pyrimidine (4-APP) on lipid peroxidation in rat liver was characterized. In 4-APP (50mg/kg)-treated rats, the amount of lipid peroxides increased from 150 to 380nmol/g of liver. The action of 4-APP on lipid peroxidation was investigated in an in vitro system containing rat liver microsomal phospholipid liposomes, NADPH, purified microsomal NADPH cytochrome c reductase, and Fe3+-ADP complexes. The addition of 4-APP resulted in the enhancement of lipid peroxidation monitored by O2 consumption and the peroxidative cleavage of unsaturated phospholipids. Superoxide dismutase, the iron chelator desferoxaminemethanesulfonate, and ceruloplasmin inhibited the 4-APP-induced lipid peroxidation. In contrast, catalase and sodium benzoate, which are known to scavenge hydrogen peroxide and hydroxyl radicals, failed to prevent phospholipid peroxidation. These findings strongly suggest that the enzymatic reduction of Fe3+-ADP-4-APP complexes by NADPH cytochrome c reductase significantly promoted lipid peroxidation in microsomal phospholipid membranes, and was probably mediated by an active iron-oxygen complex.
Probucol, a lipid-lowering drug, is transported in plasma primarily in the lipoproteins, mainly low density lipoproteins (LDL). This study examines the effect of probucol on the physical and chemical properties of LDL and high density lipoproteins (HDL) in three groups of subjects: 14 healthy normolipidemic subjects (control group), 15 untreated patients with type IIa hyperlipoproteinemia (HC group), and 16 patients treated with probucol 500mg twice daily (HC-Prob group). The lipoprotein fluidity parameters (fluorescence anisotropy and flow activation energy) were assessed by diphenyl hexatriene (DPH) fluorescence polarization. Compared with control group lipoproteins, untreated HC group LDL exhibited a lower triglyceride content and a significantly higher cholesteryl ester (CE) to triglyceride (TG) ratio. There was no difference in HDL chemical composition between the two groups. DPH fluorescence anisotropy and flow activation energy were significantly increased in both LDL and HDL from untreated hypercholesterolemic patients. Major modifications in the chemical composition and physical properties of lipoproteins were noted after probucol treatment. The triglyceride content was increased and the CE/TG ratio was decreased in both LDL and HDL. DPH fluorescence anisotropy and flow activation energy were significantly lower in LDL and HDL from treated HC patients than in those from untreated patients. Lipoproteins from the HC-Prob group were found to have the same fluidity parameters as the control group particles. The modifications of physical and chemical properties of LDL and HDL after the drug treatment can be related to the presence of probucol inside of the lipoprotein particles, which modifications in turn lead to alterations in the structure and metabolic behavior of the particles.
Serum vitamin A and its carrier proteins, retinol-binding protein and prealbumin, were measured in 41 colorectal cancer patients, 40 patients with benign colorectal disease, and 31 healthy subjects. Serum samples were obtained from the Mayo Clinic (NCI-Serum diagnostic Bank, Rochester, MN). The values for each of the three parameters were significantly lower in colorectal cancer patients than in healthy subjects but were littele different from the values seen in patients with benign disease.
To test whether monocyte-derived macrophages obtained from diabetic patients with hypertriglyceridemia have an altered manner of lipid accumulation, we separated monocytes from 7 healthy control subjects, 9 diabetic normolipidemic patients, 9 diabetic hypertriglyceridemic patients, and 7 nondiabetic hypertriglyceridemic patients. The monocytes of each group were modulated to macrophages under two different culture systems using either autologous serum or heterologous normal non-diabetic non-hypertriglyceridemic serum in the culture medium. The cells transformed better in the healthy serum system. In this system, the [14C] oleate incorporation into cholesterol oleate by macrophages derived from the diabetic group (0.30±0.11nmol/mg protein/12h (mean±SD), p<0.05) and diabetic-hypertriglyceridemic group (0.40±0.12nmol/mg protein/12h, p<0.005) was significantly higher than that by macrophages derived from the hypertriglyceridemic group (0.21±0.16nmol/mg protein/12h) and healthy group (0.22±0.02nmol/mg protein/12h). With the autologous serum system, similar results were obtained, although there was more fluctuation of the values within a group. When the degradation of labeled lipoproteins in the macrophages was determined concomitantly, the macrophages from all four groups degraded 125I-acetyl LDL equally; however the rates of 125I-labeled “hypertriglyceridemic” VLDL degradation were significantly lower by macrophages from the diabetic group (8.9±3.5μg/mg protein/12h, p<0.05) and the diabetic-hypertriglyceridemic group (8.1±1.2μg/mg protein/12h, p<0.01) than by those from healthy group (12.4±2.6μg/mg protein/12h) and hypertriglyceridemic group (11.4±3.7μg/mg protein/12h) grown in the healthy serum culture system. Macrophages from diabetic patients may have accelerated cholesterol esterification because of the lowered VLDL influx. The increase was strongly linked with the diabetic condition and was not unique to the diabetic-hypertriglyceridemic patients. This trend may accelerate atherosclerosis in diabetic hypertriglyceridemia when an atherogenic factor specific to hypertriglyceridemia coexists.