Trypsin bound to Sephadex beads: A tool for neuronal cell dissociation

Trypsin covalently bound to Sephadex beads was used for the histolytic dissociation of neuronal cell bodies from rat brain. In contrast to unbound trypsin, the bound enzyme did not bind to the isolated cells. The histolytic efficiency of the bound enzyme, measured in terms of cell yields, was approx. 100-fold greater than the free enzyme.     

Specific binding of bovine immunoglobulins to Sepharose and Sephadex

Bacteriophage T5 BglII/HindIII DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAs^Ser (tRNA^Ser₁ and tRNA^Ser₂) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non-basepaired nucleotides in the joinings of stem ‘b’ with stems ‘a’ and ‘c’.     

Studies of the distribution of proteins bound to CNBr-activated Sepharose 6B at the electron-microscopic level.

Proteins were covalently attached to Sepharose by the CNBr method. Their distribution across the carrier beads was studied at the electron microscopic level. The approach has been to ferritinstain and to section the gel beads. Ferritin was either coupled directly to the polysaccharide backbone of the carrier or conjugated with pure rabbit anti-aminopeptidase in order to visualize covalently bound leucine aminopeptidase by the immunferritin technique. The results corroborate earlier fluorescence microscopic findings of a uniform protein distribution, provided that a number of conditions are fulfilled.     

Mechanism of activation of Sepharose and Sephadex by cyanogen bromide

The mechanism of CNBr activation of polysaccharide resins like Sepharose and Sephadex has been elucidated using recently published analytical procedures for the determination of cyanate esters and imido carbonates. It was found that on agarose-based resins coupling of ligand occurs predominantly via cyanate esters, and not via imidocarbonates as in the case of Sephadex. This explains the different behaviours of Sepharose and Sephadex during CNBr activation.     

Solid-phase iodination: in vitro labeling with 125I of proteins bound to nitrocellulose and substituted Sepharose

A solid-phase iodination method is described which employs either nitrocellulose paper, phenyl- and octyl-Sepharose beads, or octyl hydroxylapatite as matrices to adsorb proteins. Nitrocellulose lends itself to cases where denaturation of the iodinated proteins due to the use of chaotropic reagents or strong acids for protein elution can be tolerated. On the other hand, substituted Sepharoses, preferably octyl-Sepharose, should be used when preservation of the biological activity of the iodinated protein molecules is required; immunoglobulins and protein A, for instance, could be recovered as functionally active molecules because they were extracted from the hydrophobic matrices under nondenaturing conditions. Both methods are advantageous if, for example, series of fractions from column chromatographies (including HPLC) are to be iodinated and subsequently analyzed by gel electrophoresis or bioassays. Furthermore, the amount of radioactive waste can be reduced considerably.     

Leucine aminopeptidase and hatching of Schistosoma mansoni eggs

Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed.     

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr-activated Sepharose and to anti-phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti-phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr-activated Sepharose or to octyl Sepharose showed reduced P_fr formation after red irradiation. The reversal to P_r with far-red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr-activated Sepharose-bound phytochrome kept in darkness and the identical sample immediately after a far-red irradiation.
The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far-red light, was measured. It was computed that the long-wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr-activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.     

Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.     

Enzymatically active subunits of Bacillus stearothermophilus enolase bound to Sepharose

The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single-point attachment. The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase. The Sepharose-bound subunit regained activity upon removal of the denaturant. It was also possible to rehydribize immobilized monomers to native octamers. Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme. Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.     

Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles.

A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually +/- 5%. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesions.     

Leucine aminopeptidase of neutrophils and lymphocytes in patients with cancer

The leucine aminopeptidase activity has been determined by using the cytochemical method of Burston and Folk in peripheral blood neutrophils and lymphocytes of 45 patients with various malignancies. Lung cancer, carcinoma of the stomach and cancer of the colon was diagnosed in 24, 16, and 5 patients, respectively. Patients with metastases showed a significantly higher activity of the enzyme if compared with that in the control group of healthy subjects and patients without metastases. The percentage of enzyme-positive lymphocytes was elevated significantly in patients with metastases whereas a total percentage of lymphocytes with regard to differential leukocyte count was diminished both in patients with and without metastases. The absolute count of neutrophils was elevated both in patients with and without metastases. The authors discuss the significance of their observation with regard to the antitumor cytotoxic effect of neutrophils and lymphocytes.     

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.     

Immobilization of flax protoplasts in agarose and alginate beads. Correlation between ionically bound cell-wall proteins and morphogenetic response.

Linum usitatissimum protoplast-derived colonies that are cultured in auxin-supplemented medium and immobilized in Ca(2+)-alginate matrix form round colonies that develop into polarized, embryo-like structures. On the other hand, protoplast-derived colonies that are immobilized in agarose do not show an organized morphogenetic response, and unique, ionically bound cell-wall protein patterns match this response. Although only slight differences in neosynthesized or total constitutive polypeptides are observed, dramatic changes in ionically bound cell-wall proteins are seen. In protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium, several basic polypeptides were strongly induced and were found tightly bound to the cell wall. In contrast, these basic proteins were found only weakly bound to the walls of protoplasts grown on agarose-solidified, auxin-containing medium or on Ca(2+)-alginate-solidified, auxin-free medium, in which they were released into the medium. Our results suggest that plant cells can perceive and respond to the adjacent extracellular matrix, since we show that the growth of flax cells on Ca(2+)-alginate in the presence of auxin-containing medium may promote the binding of specific proteins to the walls. This establishes a direct correlation of an embryo-like morphogenesis with ionically bound cell-wall basic proteins in flax protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium.     

Application of spectrophotometric methods to the determination of protein bound to agarose beads.

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates resulting in a decline in fluorescence. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates in this assay procedure. The activity of 1 μg chymotrypsin or trypsin and 100 ng pepsin could be easily detected by this method within 4 to 8 min depending upon the protein substrate. The digestive enzymes and bromelain exhibited activity against most if not all six of the protein substrates used. In contrast, the extract from germinating pumpkin seeds exhibited significant activity only against pumpkin seed globulin, with maximal activity at pH 7.4. Compared with the assay method for proteolytic activity utilizing ninhydrin analysis of the reaction products, this method was at least 10 times more rapid and gave significant detectable activity with much lower quantities of proteolytic enzyme.     

Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.     

Leucine aminopeptidase activity in the digestive tract of perch, Perca fluviatilis L.

The distribution of the enzyme leucine aminopeptidase (LAP, EC 3.4.1.1) in the digestive tract of perch, Perca fluviatilis L., was investigated histochemically. The enzyme was present in the mucosa of the entire intestine and was absent in the oesophagus, stomach and pyloric sphincter. In the intestine, the enzyme was localized in the supranuclear cytoplasm of the columnar cells and was strongest in the brush border area. Enzyme activity was also present in the lumen of the intestine. The activity of the enzyme in the intestine decreased slightly towards the rectum. In perch, the final digestion of peptides and aminopeptides is both extracellular and intracellular and takes place along the entire length of the intestine.