Effects of GTP on binding of (3H) glucagon to receptors in rat hepatic plasma membranes.

In this study, we report the preparation of [3H]glucagon and its characteristics of binding to receptors in the rat liver plasma membrane. Binding of the labeled hormone is optimal at pH 7.0. In the absence of GTP, [3H]glucagon binding to receptors is slow and the time of equilibration is inversely proportional to the hormone concentration. In the presence of GTP, equilibrium is reached within 30 s regardless of hormone levels, and the kinetics of binding are in accord with the kinetics of activation of adenylate cyclase by native glucagon in the presence of the nucleotide. Equilibrium binding measurements indicate that, in the absence of GTP, the binding isotherm is sigmoidal with an apparent Kd of 2 nM. The addition of GTP results in a complex binding isotherm with about 90% of the binding sites having a considerably lower apparent dissociation constant (greater than 10 nM) and a small population of sites having high affinity for the hormone. The binding properties of [3H]glucagon are compared with those of 125I-glucagon, and the implications of the actions of GTP on glucagon binding are discussed in relation to the overall regulation of adenylate cyclase by hormone and the nucleotide.     

Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

Specific fluorescent bands on chromosomes produced by acridine orange after prestaining with base specific non-fluorescent DNA ligands

Metaphase chromosomes stained with acridine orange exhibit uniform yellow-green fluorescence. Chromosome preparations treated with the non-fluorescent A-T specific antibiotic distamycin A prior to acridine orange staining exhibit longitudinal fluorescent banding patterns similar to those produced by a number of fluorescent R-band techniques. Similarly, chromosome preparations treated with the non-fluorescent G-C specific antibiotic actinomycin D followed by acridine orange staining exhibit Hoechst-type banding patterns. Interactions of various ligand-DNA combinations in solution indicate that the base pair specific antibiotics induce banding patterns by selectively altering acridine orange binding sites in chromosomal regions rich in the particular base pair for which the antibiotic exhibits specificity.     

Sister chromatid exchanges in human cells and Chinese hamster cells: Evidence that the rate of sister chromatid exchanges is a function of ploidy

The frequency of sister chromatid exchanges (SCE) in the chromosomes of the diploidy and polyploidy of Chinese hamster cells and human cells has been studied using BUdR-DAPI (bromodeoxyuridine, 4′-6-diamidino-2-phenylindol) fluorescence. The rate of SCEs per cell under constant control conditions is in proportion to the ploidy levels. In addition, the frequency of SCEs observed in a given human chromosome (nos. 1) is also directly proportional to the number of such chromosomes presented in the cells. The mean of SCEs in human chromosome numbers 1 is very similar (0.46–0.48) for diploid, triploid, and tetraploid cells. The results suggest that the rate of SCEs is a function of cellular ploidy levels.     

The cholesterol turnover, synthesis, and absorption in two sisters with familial hypercholesterolemia (type IIa).

To explore the mechanisms of the profound plasma cholesterol elevations in familial homozygous hypercholesterolemia (type IIa), cholesterol turnover, sterol balance, cholesterol absorption, and low density lipoprotein studies were carried out under controlled dietary conditions in two sisters (aged 13 and 16). Cholesterol turnover was prolonged. The half-life of the first exponential of the plasma cholesterol specific activity decay curve was double that of normal adults. The rate constants for the removal of cholesterol from pool A (KAA = 0.0652) and for the excretion of cholesterol from the system (Kaa = 0.0197) were less than half of normal. The production rates of cholesterol were low, only 6.30 and 6.86 mg/kg per day as measured by cholesterol turnover and sterol balance techniques, respectively. Fecal neutral steroid and bile acid excretion were 5.22 and 1.64 mg/kg per day, which is remarkably low in comparison to those of normal and heterozygous children. Cholesterol absorption was within the upper limit of the values reported for normal adults. THE HDL cholesterol values were extremely low (27 mg/dl) in contrast to profoundly elevated LDL levels. The fractional catabolic rate of LDL (0.127 per day) and the rate of synthesis and catabolism of apo-LDL (15 mg/kg per day) were low in comparison to previously reported values in homozygotes. These composite data indicated that the definable metabolic defects of these two sisters with homozygous familial hypercholesterolemia were the sluggish clearance of cholesterol from the body coupled with low total body synthesis of cholesterol.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

In vitro microwave effects on human neutrophil precursor cells (CFU-C)

Human marrow cells were irradiated with 2450-MHz CW microwaves in a fluid-filled waveguide irradiation system. Cell exposure was conducted by placing a marrow cell suspension in 20-μl glass microcapillary tubes that were positioned in the exposure chamber, and irradiated at power densities from 31 to 1,000 mW/cm² (with corresponding specific absorption rates of 62 to 2,000 mW/g) for 15 minutes. The temperature of the sample was maintained at a fixed point. Sham-irradiated (SI) and microwave-irradiated (MWI) cells were cultured in a methylcellulose culture system for neutrophil colony proliferation. There was no reduction in neutrophil colony number on days 6–7 or 12–14 in cells exposed at 31 or 62 mW/cm², but as the power density was increased to 1,000 mW/cm², there was a reduction in colony number of MWI cells compared with SI cells. The microwave interaction with the human neutrophil colony-forming cells was apparently not related to temperature rise, or to the state of cell cycle, and was irreversible.     

Size characteristics of human leucocyte interferon under reducing and non-reducing conditions.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.     

A Squash Technique for Studying the Cytology of Maize Endosperm and Other Tissues

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. the procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromsomes. with a slight modification the technique has been applied successfully to root tips and other tissues.