High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with ³²P_i. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of ³²P) and not sedimentable (supernatant) material. About 90% of ³²P in the supernatant fraction was sedimentable at 100,000 g (disperse chromatin). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of ³²P. The ³²P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the ³²P-labeled nuclear proteins into 12 major bands in the M_r range of 12,000–120,000. The incorporation of ³²P into most bands reached a steady-state within 5–10 min of incubation with ³²P_i and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an M_r 95,000 phosphoprotein. Two of the fastest moving ³²P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving ³²P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.     

Comparative studies of the binding of some ligands to human serum albumin non-covalently attached to immobilized Cibacron Blue, or covalently immobilized on Sepharose, by column affinity chromatography.

A comparative study of the ligand-binding properties of human serum albumin was performed by the technique of affinity chromatography with the protein attached to immobilized Cibacron Blue F3GA (Blue Sepharose), or covalently immobilized on Sepharose. The binding strength of octanoate, decanoate and dodecanoate is much weaker when human serum albumin is attached to immobilized Cibacron Blue, indicating that the binding sites for fatty acids are involved in the attachment of human serum albumin to immobilized Cibacron Blue. The results revealed additional alterations of the ligand binding when human serum albumin was attached to immobilized Cibacron Blue, involving sites outside of the binding domains of fatty acids. Thus the stereoselective binding of L-tryptophan was abolished, and the resolution of the warfarin enantiomers was impaired. However, the binding strength of warfarin and salicylic acid was rather close to the values observed with human serum albumin covalently immobilized on Sepharose. It is suggested that the availability of the binding sites for L-tryptophan, warfarin and salicylic acid is partially blocked by the complex between albumin and the dye without direct participation in the complex-formation. An alternative interpretation involves an allosteric mechanism brought about by complex-formation between serum albumin and the immobilized Cibacron Blue.     

Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide

To a Sepharose gel the pyridine nucleotide NAD(H) has been bound using dicyclohexyl carbodiimide. In order to improve the steric availability of the nucleotide for added soluble enzymes such as dehydrogenases, a spacer molecule, ε-amino caproic acid, was inserted between the carbohydrate matrix and the nucleotide. The obtained preparation contained 56 μmoles NAD⁺/g dry polymer. The obtained matrix-bound NAD(H) was accepted as coenzyme by added lactate dehydrogenase. These preparations were still active after storage for several weeks at 4° C and could be used repeatedly without loss of activity. This represents the first necessary step taken in the preparation of compact closed systems consisting of “enzyme–coenzyme–coenzyme-regenerating enzyme” bound to individual polymer beads; such systems eliminate the need for continuous coenzyme addition.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

Peptides with NH2-terminal tryptophan in the adenohypophysis

Summary In the mammalian pituitary formaldehyde-ozone treatment induces strong fluorescence in the cells of the pars intermedia and moderate to strong fluorescence in numerous cells of the pars distalis. Maximum excitation is at 370–375 nm and maximum emission at 495–505 nm. The properties of the cellular fluorescence are indistinguishable from those of tryptamine or peptides with NH₂-terminal tryptophan. From chemical analysis such peptides seem to occur abundantly in the mammalian pituitary. The concentration of these peptides agrees very well with the number and fluorescence intensity of the cells in all species studied. Furthermore, the tryptophyl peptides in the various parts of the pig pituitary have a distribution quite parallel to that of the fluorescent cells. As we have failed to detect tryptamine in the pituitary, we conclude that the formaldehyde-ozone-induced fluorescence in the adenohypophysis reflects the presence of tryptophyl peptides.This study was supported by grants from the Swedish Medical Research Council (04X-1007; 04X-3764), the Ford Foundation, Harald and Greta Jeanssons stiftelse and Riksföreningen mot Cancer (660-K73-01X).For brevity occasionally referred to as tryptophyl peptides.     

Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig a chemical,fluorescence histochemical,and electron microscopic study

Summary The urethra and prostate of the guinea-pig contain at least two types of endocrine-like cells in the epithelium. The predominant type is argentaffin and stores 5-hydroxytryptamine. Treatment with reserpine or a dopa decarboxylase inhibitor markedly reduces the 5-hydroxytryptamine content of this cell type. The other less numerous cell type, which is argyrophil but not argentaffin, is devoid of 5-hydroxytryptamine but can be induced to store dopamine if supplied with dopa. Both cell types occur disseminated in the urethral epithelium, whilst only the argyrophyl, non-argentaffin cell type devoid of 5-hydroxytryptamine is found in the prostate. At the ultrastructural level the argentaffin cell type contains numerous electron-dense cytoplasmic 800–1000 Å granules. These granules are argentaffin, suggesting that they are the storage site for 5-hydroxytryptamine. The cells sometimes reach the urethral lumen via a narrow neck, the apex being endowed with microvilli. This arrangement suggests that the cells are capable of responding to stimuli in the urethral lumen. Preliminary attempts to test the effect of depriving or loading guinea-pigs with water failed to induced changes in the 5-hydroxytryptamine content of the urethral endocrine-like cells.     

The copolymeric structure of pig skin dermatan sulphate. Isolation and characterization of l-idurono-sulphate-containing oligosaccharides from copolymeric chains

Dermatan sulphate was degraded by testicular hyaluronidase and an oversulphated fraction was isolated by ion-exchange chromatography. This preparation, which contained fairly long segments derived from the non-reducing terminal portion of the molecule, was subjected to periodate oxidation under acidic conditions. The oxidized iduronic acid residues were cleaved by reduction-hydrolysis (Smith-degradation) (Fransson & Carlstedt, 1974) or by alkaline elimination. The oligosaccharides so obtained contained both GlcUA (glucuronic acid) and IdUA-SO(4) (sulphated iduronic acid) residues. Copolymeric oligosaccharides obtained after alkaline elimination were cleaved by chondroitinase-AC into disaccharide and higher oligosaccharides. Since the corresponding oligosaccharides obtained by Smith-degradation were unaffected by this enzyme, it was concluded that the carbohydrate sequences were GalNAc-(IdUA-GalNAc)(n)-GlcUA-GalNAc. The iduronic acid-containing sequences were resistant to digestion with chondroitinase-ABC. It was demonstrated that the presence of unsulphated N-acetylgalactosamine residues in these sequences could be responsible for the observed effect. This information was obtained in an indirect way. Chemically desulphated dermatan sulphate was found to be a poor substrate for the chondroitinase-ABC enzyme. Moreover, digestion with chondroitinase-ABC of chondroitinase-AC-degraded dermatan sulphate released periodate-resistant iduronic acid-containing oligosaccharides. It is concluded that copolymeric sequences of the following structure are present in pig skin dermatan sulphate: [Formula: see text] N-acetylgalactosamine moieties surrounding IdUA-SO(4) residues are unsulphated to a large extent.