Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

Analysis of guanase by agarose gel electrophoresis and activity staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato

Intensely pigmented and spherical vesicles (anthocyanoplasts) were found in anthocyanin-containing cells of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin synthesis began to first occur 24–48 h after exposure to light, and then numerous small red vesicles were detected under a microscope. The frequency of anthocyanoplast-containing cells rapidly increased to finally about 80% of the total cultured cells after 5 days of irradiation. Fully developed anthocyanoplasts reached 10–15 m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured cells. 2,4-D also inhibited anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Interaction of digitonin and its analogs with membrane cholesterol

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic study of exogenously administered ß-endorphin using a rapid radioreceptor assay in rats

A rapid radioreceptor assay for measuring ß-endorphin (ß-EP) in unextracted serum has been developed. The method is based upon the inhibition by ß-EP of ³H-naloxone binding to the specific receptors on rat brain membranes, prepared in a stable form of pellets. Effect of serum on the assay was minimized by adding pooled serum to the equal dilution of total serum in the assay mixture. Pharmacokinetic analysis of pharmacologically active ß-EP equivalents (ß-EP eq.) in rats was performed using this method. The serum disappearance of ß-EP eq. after iv administration followed a biexponential decline and pharmacokinetic parameters were calculated by a two-compartment open model. The half-lives of α-phase and ß-phase were 2.6 ± 0.5 min and 6.2 ± 1.6 hr (mean ± SE; n=6), respectively. The volume of the central compartment (V₁) and that of steady-state (Vd_ss) were 67 ± 16 and 480 ± 75 ml/kg (mean ± SE; n=6), respectively. The total body serum clearance (CL_tot) was 2.1 ± 0.9 ml/min/kg (mean ± SE; n=6). The serum disappearance curve of ß-EP eq. obtained in the present study was similar to that previously reported by Houghten et al. (Proc. Natl. Acad. Sci. U.S.A. $\text{77}$, 4588–4591 (1980)), in which the disapperance of total radioactivity of tritiated ß-EP in rats was examined.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.