Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.     

Unimpaired formation of hormone-stimulated inositol trisphosphate in human mesangial cells under hyperglycemic conditions.

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grown under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (Km values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally, hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-trisphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol trisphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Fractal analysis of protein chain conformation

This paper presents a simple practical method for characterizing conformation of protein chains. A single number Df, as the fractal dimension, is assigned to each chain. Df = Ln(N)/Ln(N.d/L), where N is the number of the amino acid residues in the chain, L and d are the total length and the planar diameter of the chain, respectively. In general, 1 less than Df less than or equal to 2, which is related to the shape of the protein chain. These values are different from those of Stapleton's group, but in agreement with computer simulations.     

High frequency differences between high and low voltage electrocorticograms in the ovine fetus

Dawes (1986) has stated that, "The difference between high and low voltage activity depends solely on the presence in the latter of higher amplitude oscillations with relatively low frequency superimposed on the low voltage components as shown by spectral analysis". We have tested the constancy of the high frequency section of the power spectrum of the electrocorticogram in 7 near-term sheep fetuses. Under sterile conditions we implanted biparietal electrodes in the dura and a ground lead subcutaneously on the scalp. Five days after the surgery, with the animal standing quietly in the laboratory, we acquired the fetal electrocorticograms. Data were acquired during several high and low voltage electrocorticographic cycles in each animal. Two hundred power spectra were obtained during high and low voltage fetal electrocortical activity and statistically analyzed by paired t-test to discern differences in power between the high and low voltage pattern at each frequency (n = 7). We found that at all frequencies between 4 and 12 cycles/s the high voltage electrocorticogram had significantly more power than the low voltage electrocorticogram (P less than 0.05). This is in accordance with the established literature. We also observed that from 17 through 24 cycles/s the low voltage electrocorticogram is significantly higher than the high voltage electrocorticogram (P less than 0.05). In this frequency range the power of the high voltage expressed as a percentage of the power of the low voltage were respectively, 80, 74, 71, 66, 64, 64, 67, 64. These differences are of considerable magnitude and may be physiologically important.     

Preliminary study of the distribution of the toxic elements As,Cd, and Hg in human hair and tissues by RNAA

In order to study the relationships between trace element concentrations of hair and internal body burdens, a radiochemical NAA technique has been used for determination of the elements As, Cd, and Hg in autopsy samples of liver, kidney-cortex, lung, and hair from 24 male persons who died by accident. High significant positive correlations were observed between the As concentration in hair and in kidney-cortex, and between Cd and Zn concentrations in kidney-cortex. The contents of Cd, both for lung and kidney-cortex, were related to the smoking habits of the subjects.