Na-phosphate Buffer Research Articles

Parathyroid cell variants may be provoked during immersion fixation.

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.

Inactivation by 6-hydroxydopamine of the cerebral factor(s) responsible for the inhibition of the autoxidation of norepinephrine in vitro

The effect of extracts from rat cerebral cortex was examined on the stability of norepinephrine-HCl (NE) in 16 mM Na-phosphate buffer, pH 7.4, at 37°C. The autoxidation products of NE were detected spectrophotometrically at 480 nm. Dialysed samples from a synaptosomal preparation and from the 100,000 g supernatant of a crude homogenate were tested. Aliquots from these preparations, in the range of 0.005–5.0 or 0.01–10.0 μg protein/ml, respectively, produced up to 80–85% inhibition of the autoxidation of 100 μM NE for a period of at least 3 h. Similar results were obtained with albumin and ovalbumin at 10- and 10 3-times higher concentrations, respectively. After the preparations were exposed to 0.1–1.0 mg 6-hydroxydopamine-HCl/mg protein for 5 min at 25°C followed by rapid dialysis, the maximal inhibitory effect was reduced to between 95% to less than 5% of control values. The percent inactivation by a given quantity of 6-hydroxydopamine (6-OHDA) was inversely related to the potency of the untreated sample. Additional observations are presented which suggest that the destruction of the antioxidant activity is caused by breakdown products of 6-OHDA reacting with nucleophilic sites of the preparation. Similar inactivating substances are expected to be formed from other autoxidizing catecholamines, although at a slower rate.

An improved method for purification of soybean mosaic virus.

The particles of soybean mosaic virus (SMV) was successfully purified without inactivation by the following improved method. The soybean leaves infected with SMV were homogenized with two volumes of 0.3M Na-phosphate buffer (pH 7.0) containing 0.01M Na-DIECA and chloroform-butanol (each 8% v/v). The crude sap was filtered through two layers of cheesecloth and centrifuged at low speed (8, 500×g, 10min). The supernatant was supplied with 6% PEG and 0.1M NaCl. After 30min of incubation at 4C, the suspension was centrifuged at low speed, the precipitate obtained was resuspended in 0.05M Na-phosphate buffer (pH 7.0) containing 0.005M Na-EDTA, and ultrasonicated for 10 to 30 seconds. The virus fraction was collected by two cycles of differential centrifugation combined with ultrasonication just before each low speed centrifugation. The virus fraction was then subjected to the sucrose density gradient centrifugation for final step of purification. The purified virus thus obtained was highly infective. It showed the maximum ultraviolet absorption at 260nm and minimum at 247nm with a slight shoulder at 290nm. The ratio of A280/A260 was 0.76, indicating the nucleotide content of about 5.5%. The yield was 4.0 to 13.5mg per 1kg of the infected leaves.

Inhibition of ascorbate autoxidation by a dialyzed, heat-denatured extract of plant tissues

Preparations obtained from various plant sources were analyzed for their effect on the autoxidation of ascorbic acid and norepinephrine. The former reaction was followed by spectro-photometric detection of ascorbic acid at 265 nm, the latter one by measuring the formation of noradrenochrome at 480 nm. Extracts were prepared from Philodendron leaves and the edible portion and seeds of green peppers ( Capsicum Annuum ). The tissues were minced, homogenized in 10 volumes of 16 mM Na-phosphate buffer pH 7.4 and centrifuged at 35,000g for 30 min. The supernatant was dialyzed in 12,000 m.w. cut-off tubing, denatured in boiling water and centrifuged at 10,000g for 10min. Aliquots (5–50 ul) of the supernatant were assayed in 5 ml 16 mM Na-phosphate buffer pH 7.4 containing 100 uM ascorbate or norepinephrine. The denatured extracts had marked dose-dependent inhibitory effect on the autoxidation of ascorbic acid, with negligible influence on the formation of noradrenochrome. EDTA inhibited both reactions. The selectiveness of the extract toward the autoxidation of ascorbic acid makes it unlikely that the inhibitory effect is based on sequestering metal-ions.

Effect of lipid composition on hydrophobic properties of liposomes

Partition behavior of liposomes of different composition in the aqueous Ficoll-Dextran biphasic system was examined. The relative hydrophobicity of the liposomes was estimated. The effect of cholesterol on the solvent interactions of lecithin and sphingomyelin liposomes was studied. It is found that the effect of phosphatidylethanolamine on the hydrophobic character of lecithin liposomes exceeds that produced by cholesterol. The relative hydrophobicity of all the liposomes examined was found to be independent of the ionic composition of the system containing varied amounts of NaCl and Na-phosphate buffer, pH 7.4. The hydrophobic character of the liposomes under study is shown to increase in the following order: phosphatidylserine less than phosphatidylethanolamine less than phosphatidylcholine less than sphingomyelin. Hydrophobic properties of the liposomes are compared to those of human red cells. It is suggested that the dependence of the hydrophobic character of membrane particles on the ionic composition of an aqueous environment can be of fundamental importance for the selective distribution of the particles in vivo.

Rapid Assay of Hypothalamic Aromatase Using High Performance Liquid Chromatography

Abstract A rapid assay for hypothalamic aromatase has been developed using HPLC. Each assay tube contained rat anterior hypothalamus homogenized in Na phosphate buffer (pH 7.0), NADPH regenerating system, unlabelled estradiol and estrone, and 10 μCi of 3H-androstenedione. After agitation at 37°C for 3 hours, the reaction mixture was extracted with CH2Cl2, partitioned between 90% methanol and hexane, and the methanol layer chromatographed on a Waters 10μ C-18 Radial-PAK column using acetonitrile/water (70:30). The estrogens were collected together and rechromatographed with THF/water (35:65). The estrone peak was collected, and the 3H-estrone produced was determined by liquid scintillation. The estrone collected from the second chromatographic step was found to be at least 95% pure by methylation and rechromatography. Using the HPLC procedure, enzymatic 3H-estrone formation was found to be linear in a time and protein dependant manner.

Protein-synthesis inhibitory protein from seeds of Luffa cylindria roem

A protein (designated as luffin) with an app. M r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate ( IC 50, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10 6 to 1/10 5 that of ricin.

Effects of detergents on lysis of Micrococcus lysodeikticus by lysozyme.

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments. None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.

Buffer-dependent variations in assay of tyrosine aminotransferase holoenzyme

Homogenization of rat liver in Hepes ( N-2-hydroxyethylpiperazine- N′-2-ethane-sulfonic acid), MOPS (2-[ N-morpholino]ethanesulfonic acid), Na phosphate, Pipes (piperazine- N, N′-bis[2-ethanesulfonic acid]), TEA (triethanolamine), TES ( N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid), Tricine ( N-tris-[hydroxymethyl]methylglycine), or Tris (tris[hydroxymethyl]aminomethane), and subsequent assay for supernatant total and holo tyrosine aminotransferase activity using these buffers yields apparent enzyme concentrations which vary depending upon the buffer composition, the ionic strength, and the fold-dilution of the supernatant. A precipitous decrease in the apparent holoenzyme concentration results from a slight dilution of the supernatant with most of the buffers. Some of the dilution effects may be due to dissociation of pyridoxal phosphate from the apoenzyme or to competition between the buffer and pyridoxal phosphate for association with the enzyme. The percentage of the apparent total enzyme which exists as holoenzyme varies from 3% for supernatant prepared in Na phosphate buffer up to 94% for that prepared in Hepes. Inactivation of total enzyme activity occurs to a similar extent resulting from incubation of liver homogenates prepared with Na phosphate, Hepes, or Pipes. The residual apparent holoenzyme activity observed when assayed in the presence of Na phosphate may be due to reaction of an enzyme other than tyrosine aminotransferase. The data provide a basis for explaining the large variation in reported percentage holoenzyme and should also serve as a warning for other holoenzyme assays which use pyridoxal phosphate as a cofactor.

Swelling of the Müller fibers in the chicken retina.

A high potassium concentration (33 meq) in the solution superfusing the isolated chicken retina causes an increase in the tissue transparency. An L-glutamate (1 mM) or L-proline (10 mM) solution has the same effect. Swelling of the Müller fibers, which have a radial position in the retina, could explain the transparency increase. This possibility was investigated in electron micrographs of retinas subjected to these treatments and fixed by freeze-substitution to preserve the water distribution in the tissue. The Müller fibers in the controls had a mean diameter of 0.22 micrometer. The fibers in retinas bathed for 2 min in the high [K+] solution were more than three times as thick (0.74 micrometer); the fibers in glutamate-treated retinas were more than twice as thick (0.49 micrometer). The fibers in the proline-treated retinas had a diameter of 0.39 micrometer. The glutamate- and proline-induced swelling may be due to a K+ release from neuronal elements, acting on the Müller fibers. The fiber swelling was postulated to be the expression of different Donnan equilibriums of fibers bathed in solutions of different K+ concentrations. The observed swelling caused by the high [K+] solution was compared with the theoretical swelling of the fiber as an ideal Donnan system, postulating permeabilities for different ions of the fiber membrane. This suggested that the high [K+] solution causes an increase in Na+ permeability in addition to the permeability of the membrane for K+, Cl-, and HCO3-. Chemical fixation with glutaraldehyde and formaldehyde in an Na-phosphate buffer yielded micrographs in which the Müller fibers of retinas treated with a high [K+] or a glutamate solution had diameters similar to those of the control preparations.

Identification of glucan synthetase glycosidically bound to amylopectin from cotton leaf starch

Starch-granule bound glucan synthetase was isolated from cotton leaves by a sucrose density technique. Starch granules were successively washed with 0.02 M Na-phosphate buffer containing 0.9% NaCl. The alkaline hydrolysate of washed materials and acid hydrolysate of protein-bound amylopectin were separated by use of Con A-Sepharose. Amylolytic hydrolysate of starch granules also was carried out with α-amylase and fractionated by chromatography on Sephadex G-25. Amylopectin was the only glucan glycosidically linked with proteins, in which glucan synthetase activity was identified. The amylose component was free from covalently bound proteins.

Structure of β-casein

The temperature and concentration dependent association of β-casein was studied by means of viscometry, gel filtration chromatography, electron microscopy, analytical ultracentrifugation and UV difference spectrophotometry. Degrees of polymerization of 12, 22 and 49 and free energies of association of –21, –23 and – 25 kJ/mole monomer were found at temperatures of 10, 15 and 20 °C respectively in 0·2 M Na phosphate buffer pH 6·7.Monomeric β-casein was not a completely random coil but became more compact with increasing temperature, due to hydrophobic interactions.

1104 CRYOACTIVATION AND ACID ACTIVATION OF AN INACTIVE RENIN IN THE NEWBORN INFANT

An inactive large molecular form of renin which can be acid and cryoactivated has been described in human amniotic fluid and plasma. The present study was conducted to determine whether the concentration of inactive renin is high in the newborn at a time when circulating active renin levels are known to be elevated, and whether inactive newborn renin can be separated from active renin by chromatography. 78 samples of newborn plasma were acidified to ph 5.5-5.7, the optimal ph for generating angiotensin I (angio I) from active renin, and treated with Bal and 8-OH quinoline to prevent formation of angiotensin 2. The samples were divided into 2 equal parts, one control for circulating angio I and one generated to angio I at 37°C for 1 hr prior to RIA. The control and generated samples were placed in an ice bath (3-4°C) for 10 hrs. The control samples contained 6.7 ± 1.0 ng/ml/hr (M and SEM) of active renin, and 14.1 ± 1.8 of cryoactivated renin (as PRA). The (37°C, 1 hr) generated samples contained 22.8 ± 1.3 ng/ml/hr of active renin and 17.7 ± 1.7 of cryoactivated renin (as PRA). Chromatography on 1M G-75 Sephadex columns eluted with 0.1M Na Phosphate buffer, ph 6.9 showed active renin in one fraction, M.W. 45,000, and cryoactivated and acid activated (ph 3.0) renin in another fraction, M.W. 67,000. Conclusions: a) active and inactive renin are both in high concentration in newborn plasma, b) cryoactivated and acid activated renin are of similar M.W. and larger than active renin.

Action of the heparin polyanion on chromatin preparations obtained in solutions of low ionic strength

DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b.

Effect of mercury on NADH and the protective role of oxalacetate

Because of the many biochemical roles of heavy metals experiments were done to examine the nature of the interaction between NADH and mercuric chloride. The comparative effects of HgCl/sub 2/ and methylmercury chloride on beef heart lactate dehydrogenase were studied spectrophotometrically following the oxidation of NADH by pyruvate. When 0.003 x 10/sup -6/ M MM was present in the 3 ml reaction mixture LDH activity was not inhibited. When the mixture contained 0.03 or 0.3 x 10/sup -6/ M MM, the enzyme activity was inhibited 11.7 and 41.7 percent compared to the control. When 0.0003 x 10/sup -6/ HGCl/sub 2/ was added to the reaction mixture in place of MM there was no inhibition of LDH activity. The enzyme was completely inhibited by 0.15 x 10/sup -6/ M HgCl/sub 2/. The addition of 0.2 x 10/sup -6/ M HgCl/sub 2/ to Na-phosphate buffer containing 0.2 x 10/sup -6/ M NAD had no effect on the normal NAD spectra. When the NADH-HgCl/sub 2/ reaction mixture was incubated 2 min prior to the addition of LDH enzyme, it was noted that the absorbance at 340 nm decreased rapidly due to interaction between NADH and HgCl/sub 2/. Various levels of other intermediate metabolitesmore » such as L-malate, succinate, tartarate and oxaloacetate or pyruvate were added to the NADH-HgCl/sub 2/ complex in the reaction mixture. Only OAA restored, in part, the absorbance at 340 nm. The data revealed that HgCl/sub 2/ inhibited LDH more than did MM, probably due to the monofunctional nature of MM, which forms one ligand (R-Hg-L), whereas HgCl/sub 2/ is bifunctional and can react with two ligands (L-Hg-L). The results indicate that OAA protects NADH by reaction with Hg/sup 2 +/. (MU)« less

Fragmentation of benzylpenicillin after interaction with the exocellular DD-carboxypeptidase-transpeptidases of Streptomyces R61 and R39.

THE killing target of penicillin in bacteria is a membrane-bound transpeptidase which catalyses peptide cross linking during wall peptidoglycan synthesis1,2. Streptomyces R61 and R39 excrete during growth DD-carboxypeptidase-transpeptidase enzymes3,4 which seem to be soluble forms of the corresponding membrane-bound transpeptidases5. The exocellular enzymes (E) convert penicillin (I) in to a chemically altered and biologically inactive compound (X)6,7. Kinetically, the simplest mechanism5 for the conversion of I into X is The first step, a rapid equilibrium process, leads to the formation of an equimolar and inactive enzyme–antibiotic complex EI. This complex isomerises into a modified complex EI* which, in turn, undergoes irreversible breakdown. If the experiment is carried out in conditions in which the enzyme is stable, the enzyme is reactivated and recovers its initial penicillin sensitivity. The breakdown of complex EI* is a slow process. At 37 °C and in 10 mM Na phosphate buffer, pH 7.0 (in which conditions the R61 enzyme is stable) the half life of the R61 enzyme–benzylpenicillin EI* complex is 80 min (ref. 7). At 37 °C and in 0.1 M Tris-HCl buffer, pH 7.7, containing 0.1 M NaCl and 0.05 M MgCl2 (in which conditions the R39 enzyme is stable), the half life of the R39 enzyme–benzylpenicillin is 4,250 min (ref. 6). As the enzyme is reactivated during the process, no enzyme is irretrievably lost on reaction with penicillin and after several cycles of inactivation and reactivation, both the enzyme and the accumulated X compound can be reisolated. This technique was applied to 14C-benzylpenicillin (benzyl labelled) and the 14C-X compound produced was isolated.

Giemsa technique for the detection of sister chromatid exchanges.

Sister chromatid exchanges are sharply demarcated in Giemsa stained metaphase preparations of Chinese hamster ovary cells and human peripheral leukocytes. Chromatids singly and doubly substituted with BrdU acquire differential Giemsa stain affinities after treatment at 88° C for 10 minutes in 1.0 M Na phosphate buffer (pH 8.0).