Sodium Chloride Gradient Research Articles

Effect of dietary phosphate on transport properties of pig renal microvillus vesicles.

Dietary phosphate manipulation results in stable adaptive changes in the transport functions of microvillus membrane vesicles isolated from pig renal cortex. When assayed under sodium gradient conditions, phosphate uptake is enhanced 200--400% in vesicles prepared from animals maintained on a low-phosphate diet (0.22%) compared to high-phosphate diet controls (0.82%). When transport is assayed in sodium preequilibrated vesicles, a 100% enhancement of phosphate uptake is demonstrable. Stimulation of phosphate uptake into low-phosphate diet vesicles after the imposition of a sodium chloride gradient is equivalent if uptake is measured at pH 6.0 or 8.0 and can be kinetically characterized as resulting from a Vmax alteration in the phosphate transport system. Microvillus membrane vesicle phosphate transport is maximally stimulated after only 2 days of dietary deprivation. Although a longer period (1 and 2 wk) of phosphate restriction does not further stimulate phosphate transport, it does result in an inhibtion of other sodium gradient-dependent transport systems (glucose, alanine).

Progesterone is conjugated in human urine

The discovery of two previously unknown progesterone conjugates in human urine is presented. Urine from near-term pregnant women was applied to an anion exchange column and then eluted with an increasing concentration gradient of sodium chloride. Aliquots of collected fractions were subjected to four different treatments: (1) immediate extraction, (2) incubation at 37°C, (3) β-glucuronidase hydrolysis and (4) acid hydrolysis. All eluent aliquots were then further purified on a thin layer chromatographic system and measured for progesterone content by radioimmunoassay. The results suggest that progesterone, a non-hydroxylated Δ 4-3 ketosteroid, occurs in at least two negatively charged conjugated forms in human urine. Comparison with elution patterns of known testosterone conjugates shows that these conjugates of progesterone travel on an anion exchange column similarly to testosterone glucuronide and testosterone sulphate. Although the exact structure of these progesterone conjugates remains to be elucidated, the property of physiological conjugation of progesterone has not previously been described. Quantitatively each of these conjugates appears to exist in amounts equal to or greater than the levels of free (unconjugated) urinary progesterone.

Separation and Partial Characterization of Chloroplast and Non-chloroplast a-Glucan Phosphorylases from Spinach Leaves

Chloroplast and non-chloroplast a-glucan phosphorylases (EC 2.4.1.1) were separated by ion-exchange chromatography on DEAE-Sephacel and identified by analytical polyacrylamide gel electrophoresis and co-electrophoresis with extracts of isolated intact chloroplasts. The non-chloroplast phosphorylase was eluated with a linear gradient of 20-60 mmol citrate/l (adjusted to pH 6.5 with NaOH), then the chloroplast phosphorylase was recovered with a linear sodium chloride gradient (0-1.2 mol NaCl/1 in 60 mmol citrate/1). In a control experiment, chromatography of a preparation from isolated intact chloroplasts produced only one phosphorylase activity peak which appeared during elution with the sodium chloride gradient. In a leaf extract, an approximately 1 : 3 ratio of chloroplast to non-chloroplast activity was observed. The apparent molecular weights determined by gel filtration were 230,000 for the chloroplast phosphorylase and 150,000 for the non-chloroplast enzyme. Non-chloroplast phosphorylase showed a high affinity to both starch and glycogen when assayed under saturating orthophosphate concentrations, whereas the chloroplast phosphorylase had an affinity to starch about 10 times lower and an even lesser affinity to glycogen. Both phosphorylases exhibited similar affinities to orthophosphate (Km 1-2 mmol/1) when assayed at saturating starch concentrations. Key words

Protein fractionation, nucleic acids, and enzymatic activity of cytoplasmic extracts from the intestinal mucosa of sheep

A method was designed to separate and study the cytoplasmic proteins of the intestinal mucosa of sheep. Specimens of mucosa from healthy sheep were extracted and analyzed for protein and nucleic acid content, and for selected enzyme activities. The proteins of this extract were separated by DEAE-cellulose chromatography. Samples of ulcerated mucosa were also extracted and analyzed for protein and nucleic acid content. Extracts from healthy mucosa contained more protein (71 ± 7 versus 52 ± 8 mg/ml) and less nucleic acid (8.5 ± 3 versus 14 ± 3 mg/ml) than extracts from ulcerated mucosa. The enzyme assay on healthy extracts showed that protease, amylase, alkaline phosphatase, transaminase, and lactase activity were present. Fractionation of the extracted proteins on DEAE-cellulose, using a Tris-HCl buffer (0.05 m, pH 9.0) and a sodium chloride gradient of 0.0–0.04 m, yielded 10 major protein fractions, of which 6 were eluted at less than 0.1 m NaCl. This system is useful for investigating changes in cytoplasmic proteins caused by drugs and diseases affecting the intestinal mucosa.

Chromatographic separation of orange pectinesterase isoenzymes on pectates with different degrees of cross-linking

A selected sodium pectate preparation, suspended in an alkaline mixture of ethanol and water, was cross-linked with epichlorohydrin. By varying the amount of epichlorohydrin in the reaction mixture, preparations were obtained with different degrees of cross-linking. The preparations were used as chromatographic media for the separation and purification of two orange pectinesterase isoenzymes. The binding of the isoenzymes was found to depend on the degrees of cross-linking of the pectate. On pectate with an intermediate degree of cross-linking (0.46) both isoenzymes were retained and subsequently eluted, with a sodium chloride gradient, as separated peaks with a 7- to 8-fold increase in specific activity. Both pectinesterase isoenzymes saponified 93% esterified pectin with a high degree of cross-linking. It is thought that the binding of these enzymes to cross-linked pectate involves biospecific affinity.

Studies on the effects of bradykinin and its fragments on the central nervous system.

Kininases were separated by DEAE-cellulose column chromatography with stepwise gradient of sodium chloride from rat whole brain. F-A which developed with 70 mM NaC1 cleaved Phe-Ser bond, F-B with 100 mM NaC1 hydrolyzed post-proline site and F-C with 150 mM NaC1 broke down bradykinin to amino acids. F-A activity was inhibited by Arg-Pro-Pro-Gly-Phe, Ser-Pro-Phe-Arg and Cly-Phe-Ser-Pro, and the two formers blocked F-B activity.

21 ‐ ARGININE ‐ A] INSULIN

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.

Zone precipitation chromatography: its use in the isolation of different collagen types.

Zone Precipitation Chromatography is a useful technique for the initial isolation of the different collagen types in their native configuration. Small quantities of collagen mixtures can be rapidly separated into different collagen types with a relatively high degree of purity, based upon stained protein patterns on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) slab gels. In the commonly used bulk salt preparative method for isolating the different collagens, 50 mg of starting material was needed. Three days were required to complete the procedure. The stained protein patterns on SDS-PAGE slab gels showed about 25% contamination with the bulk purified Type III fraction and 20% contamination with the bulk purified type AB collagen. With Zone Precipitation Chromatography 5 mg of starting material was used and in less than 4 hours the mixture was separated with Types III and AB fractions showing less than 10% contamination from other collagen types. The technique is patterned after the Zone Precipitation method reported by Porath seventeen years ago and utilizes a step-wise sodium chloride gradient to precipitate and redissolve the collagens, eluting from the interbead spaces of a molecular sieve column.

Oligonucleotide inhibitor of protein synthesis made in extracts of interferon-treated chick embryo cells: Comparison with the mouse low molecular weight inhibitor

Cytoplasmic extracts of interferon-treated primary chick embryo cells contain an enzyme activity that synthesized an inhibitor of chick cell-free protein synthesis. The same activity was detected in extracts of cells treated with mock preparations of interferon, but at <0.3% of the level found in interferon-treated cell extracts. The enzyme was activated by double-stranded RNA and could be isolated by binding to columns of poly(I)-poly(C)-agarose. In the column-bound state, the enzyme reacted with ATP to synthesize the inhibitor, which could then be continuously eluted from the column. The inhibitor was purified and its structure and function were compared with those of the low molecular weight inhibitor of protein synthesis made by an enzyme from interferon-treated mouse L cells. The avian and mammalian inhibitors comigrated on thin layers of polyethyleneimine-cellulose during chromatography in three different solvent systems, and they coeluted as a series of peaks from columns of DEAE-cellulose during sodium chloride gradient elution. Digestion with bacterial alkaline phosphatase or snake venom phosphodiesterase yielded products that similarly comigrated. Functionally, the two inhibitors were interchangeable: both inhibited protein synthesis in extracts of mammalian and avian cells, producing 50% inhibition at a concentration of about 0.3 nM (AMP equivalents). We conclude that the chick cell-derived oligonucleotide inhibitor has a structure that is closely related or identical to that of the inhibitor made in the mouse system, and that both preparations inhibit cell-free protein synthesis in a non-species-specific manner.

Sugar transport by brush border membrane vesicles isolated from human small intestine.

Uptake of D- and L-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake of D-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both, D- and L-glucose was inhibited by phlorizin. Uptake of D-fructose into the brush border vesicles was not stimulated by sodium. In the presence of a sodium gradient D-glucose was taken up 5 times faster than L-glucose. The amount of D-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount of D-glucose taken up at equilibrium (over-shoot). D-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradient D-glucose transport was increased by the nearly impermeable anion sulfate as compared with uptake of D-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodium coupled non-electrolyte transport.

The preparation of pure salt-free nicotinamide coenzymes

A simple procedure is described for preparing NAD +, NADP +, and their analogs in a salt-free chromatographically pure state. The previous standard method of purification by elution from DEAE-cellulose with a phosphate gradient is unsatisfactory for this purpose, because subsequent gel filtration does not separate the coenzymes from phosphate. Good results are obtained, however, with a gradient of sodium chloride, which can be adequately separated from nucleotide coenzymes on Sephadex G-10. NAD + prepared by this procedure, lyophilized, and stored for 3 months showed negligible deterioration. The problem of peak splitting of NAD + and NADP + in phosphate on Sephadex G-10 is discussed in relation to recent similar observations with ATP and formate [Bernofsky, C. (1975) Anal. Biochem. 68, 311–315].

Highly purified neurophysin proteins: Method of isolation based on their acidic properties

The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent p I value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.

Polymerization of penicillins : IV. Separation, isolation and characterization of ampicillin polymers formed in aqueous solution

Polymeric substances formed from amplicillin sodium in aqueous solution have been separated according to charge by anion-exchange chromatography on a column of DEAE-Sephadex A-25 using a linear sodium chloride gradient at a constant pH of 7.4. The separated materials, ranging in size from the monomer to the octamer, have been characterized by various functional-group analyses. A homogeneous well-defined ampicillin dimer, tetramer and hexamer have been isolated from the effluent in high yields. The separation and isolation of the ampicillin polymers permit a conclusive establishment of their structures, which in turn has confirmed previous suggestions that the polymers are formed through a chain process involving aminolysis of ampicillin at the β-lactam carbonyl moiety by the amino group of the side-chain of a second ampicillin molecule.

Isolation and Characterization of Four Peptide Hydrolases from the Cytosol of Rat Intestinal Mucosa

The high speed supernatant fluid prepared from rat intestinal mucosa was subjected to ion-exchange chromatography on diethlaminoethyl-cellulose eluted with a linear gradient of sodium chloride (0 to 0.27 M). Assay of eluted fractions for Phe-Gly hydrolase activity revealed four distinct peaks of enzyme activity. These cytosol enzymes have been designated I, II, III, and IV in order of their elution from the column. Examination of the substrate specificity of the four enzymes by use of 20 mM peptide concentrations indicated the most discriminating substrates for the four enzymes were Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide, respectively. The mean distribution of the recovered peptide hydrolase activities against these substrates among the four enzymes I, II, III, and IV was 96.1, 1.4, 1.7, and 0.8%, respectively, for Leu-Gly-Gly; 0.6, 96.4, 2.4, and 0.6% for His-Met; 0, 0, 95.8, and 4.2% for Ser-Phe; and 20.8, 19.8, 5.6, and 53.8% for leucine amide. Ion-exchange chromatography resulted in increases in specific activity of 19-, 19-, 46-, and 3.5-fold for enzymes I, II, III, and IV, respectively. The activity of all four enzymes, but especially III and IV, were stabilized by the presence of 150 muM dithioerythritol. Activity of each of the four enzymes was decreased 79 to 100% by 1mM ethylenediaminetetraacetate, HgCl2, 1, 10-phenanthroline, or 0.5 mM p-hydroxymercuribenzoate, except that the activity of enzyme I was decreased only 15% by ethylenediaminetetraacetate. No significant activation of the partially purified enzymes occurred in the presence of 500 muM Zn++, Co++, or Mg++. The four enzymes exhibited distinct pH profiles with optima at 7.5, 7.5, 8.5, and 8.0 for enzymes I, II, III, and IV, respectively. Molecular weights of the four enzymes determined by gel filtration on Sephadex G-200 were 58,500, 74,000, 97,500, and 113,000, respectively. All four enzymes lost more than 85% of their activity after 1 hr at temperatures of 50 degrees C or higher in sodium phosphate buffer, pH 7.0. The Km values determined with the most specific substrates for each enzyme were 0.76, 0.44, 3.82, and 8.3 mM for enzymes I, II, III, and IV, respectively. Recent evidence suggests that a significant amount of some small peptides are absorbed intact and hydrolyzed by cytosol peptide hydrolases. Adequate understanding of the function and control of these intracellular enzymes requires knowledge of the characteristics and substrates specificity of individual enzymes. The study described here demonstrates the presence of at least four cytosol peptide hydrolases with distinct substrate specificities. Substrates almost exclusively hydrolyzed by each of three of the enzymes, and therefore suitable for assay of each of these enzymes in the presence of the others, have been identified.

Synthesis of [a-2-alphagamma-diaminobutyric acid, a-19-glutamic acid] sheep insulin.

An analogue of sheep insulin which differs from the parent molecule in that the amino-acid residues isoleucine and tyrosine at positions 2 and 19 in the A chain are replaced by αγ-diaminobutyric acid and glutamic acid, respectively, has been synthesized. For this purpose [A2bu2,Glu19] A chain S-suphonate was synthesized by the fragment condensation approach and isolated in purified form. Conversion of the latter into the thiol form and interaction with the S-sulphonated B chain of bovine (sheep) insulin yielded [A-A2bu2,GIu19] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. This analogue, tested by the mouse convulsion assay method and in doses at least 19-fold higher than those normally used for insulin assay, was inactive. It was also inactive when tested by the radioimmunoassay method. Thus the A2 and/or A19 residues must be involved in the expression of the biological activity of insulin.

Purification of human alpha-1-fetoprotein by ammonium sulphate gradient solubilization: development of reagents for radioimmunoassay

Alpha-1-fetoprotein (AFP) was purified on a preparative scale from human cord serum and used to develop antiserum to AFP. Cord serum was applied to a QAE-Sephadex A-50 column. Elution was with an ascending sodium chloride gradient, and the AFP fraction (eightfold enrichment) after dialysis was mixed with Celite 545. The mixture was washed twice with 60% saturated ammonium sulfate to preferentially remove albumin. The washed product was equilibrated with 80% saturated ammonium sulfate, packed in a column, and eluted with a descending ammonium sulfate gradient to renove more albumin. After lyophilization the AFP fraction (one hundred- fold enrichment) was passed through a Sephadex G-200 column to remove high molecular weight proteins. The AFP rich fraction was dialyzed and then passed through an immunoadsorbent column prepared from Sepharose 4-B and rabbit anti-normal human serum. The product was 68% AFP and represented 39% recovery and greater than three hundredfold enrichment of the AFP in the cord serum. Material from the ammonium sulfate fractionation step was used as immunogen for the production of antisera in rabbits. The antisera were subsequently made monospecific by absorbing out antibodies to normal male plasma.

Synthesis of two biologically active insulin analogues with modifications at the N-terminal and N- and C-terminal amino acid residues.

The synthesis and isolation in purified form of two analogues of insulin is described. [21-Isoasparagine-A] ([Iasn21-A]) insulin differs from the parent molecule in that the amino acid residue, asparagine, found at the C terminus of the A chain (A21) has been replaced by isoasparagine. [Sar1, Iasn21-A] insulin differs from insulin in that both the A1 and A21 amino acid residues, glycine and asparagine, have been substituted by sarcosine and isoasparagine, respectively. The synthesis of these analogues followed the pattern employed in this laboratory for the synthesis of insulin and its analogues. The S-sulfonated derivatives of the A chain analogues were chemically synthesized, converted to their sulfhydryl forms, and then combined with the S-sulfonated B chain to produce the respective insulin analogues. Isolation of the insulin analogues from the combination mixtures was effected by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. By the mouse convulsion assay method [Iasn21-A]insulin possessed a potency of 21 IU/mg and [Sar1, Iasn21-A] insulin 15 IU/mg. The radioimmunoassay method gave values of 16 IU/mg for the former and 7IU/mg for the altter analogue. The natural hormone has a potency of 23-25 IU/mg by both assay methods. These data indicate that the alpha- and beta-carboxyl groups of the A21 amino acid residue are nearly equivalent in terms of their contribution to the expression of the biological activity of insulin. Furthermore, these data strengthen the speculation (Cosmatos, A., Johnson, S., Breier, B., and Katsoyannis, P. G. (1975), J. Chem. Soc. Perkin Trans. 1, 2157) that the change in the relative positive charge at the N-terminal amino acid residue of the A chain is responsible for the considerable decrease in the immunoreactivity observed in such modified insulins.

Synthesis of human [9-leucine-B] Insulin.

The synthesis and isolation in purified form of [Leu9-B]insulin, a biologically active analogue of human insulin, is described. This analogue differs from the parent molecule in that the polar residue, serine, occupying position 9 in the B chain and located on the outside of the insulin molecule, has been replaced with the hydrophobic leucine residue. For the synthesis of this analogue the [Leu9]B chain of human insulin was chemically synthesized by the fragment condensation approach and isolated in the S-sulfonated form. Combination of this compound with the sulfhydryl form of human A chain afforded [Leu9-B]insulin. Separation of this analogue from the combination mixture and isolation as the hydrochloride in purified form were accomplished by chromatography on a carboxymethylcellulose column with an acetate buffer (pH 3.3) and an exponential sodium chloride gradient. [Leu9-B]Insulin possesses a potency of 13-14 IU/mg when assayed by the mouse convulsion method and 11-12 IU/mg by the radioimmunoassay method as compared to 23-25 IU/mg possessed by the natural hormone.

NONDIALYZABLE SULFATED SIALOGLYCOPEPTIDE FRACTIONS DERIVED FROM BOVINE HEIFER BRAIN GLYCOPROTEINS. ISOLATION, CHARACTERIZATTON AND CARBOHYDRATE‐PEPTIDE LINKAGE STUDIES1

Abstract—The nondialyzable sialoglycopeptides dcrived from defatted, protease digested whole boyine heifer brain tissue were isolated and characterized. The partially purified bovine heifer brain tissue were isolated and characterized. The partially purified sialoglycopeptide mixtures contained 7‐8% ester sulfate. The amount of nonsulfated material present was of the order of 15%. The total mixture was fractionated by anion exchange chromatography on AG1‐X2(CI‐) using a sodium chloride gradient to yield twelve fractions all of which contained ester sulfate to varying degrees (5.6‐21.9%).The molecular size of the fractions ranged from molecular weights of 10,400‐12,500. The molar sulfate/galactose plus glucosamine ratios ranged from 0.3 to 1.1. Carbohydrate peptide linkage studies of the fractions by treatment with 0.2 M‐NaOH‐O.3 M‐NaBH4 led to the destruction of a portion of the threonine, serine and galactosamine and the appearance in acid hydrolysates of α‐amino‐n‐butyric acid and galactosaminitol with an increase in alanine. Partial acid hydrolysis of the most abundant sialoglycopeptide liberated detectable amounts of 2‐acetarnido‐1‐(L‐4‐aspartamido‐)‐1,2‐dideoxy‐β‐D‐ glucose. These results indicated that the GalNac at the reducing end of the alkali labile carbohydrate prosthetic groups were linked O‐glycosidically to thc hydroxyl groups of threonine and serine and the alkali‐stable region of the principal sialoglycopeptide involves N‐acetylglycosaminyl‐asparagine linkages. Mild acid hydrolysis of the alkali‐stable portion of the sulfated fractions indicated that the ester sulfate was confined to the alkali‐stable region and exists as galactose 6‐O‐sulfate and N‐acetylglucosa‐ mine 6‐O‐sulfate, structural fcatures similar to those present in rat brain glycopcptides (Markgolis &amp; Margolis, 1970; Margoliset al.; 1972). Fucose and ester sulfate were confined to the alkali‐stable region while mannose was located in both the alkali‐labilc and alkali‐stable regions of the sialoglyco‐peptide fractions.

Chromatography of hemoglobins on cm-cellulose with bis-tris and sodium chloride developers

CM-Cellulose as an ion-exchange medium with Bis-tris as buffer and a gradient of sodium chloride provides a versatile system for the chromatography of hemoglobins. Changes in pH, Bis-tris concentration, and slope of the sodium chloride gradient provide means for markedly altering chromatographic behavior for special separations. Examples are given of the application of the method to normal samples and to those with hemoglobinopathies.