Preparation of biologically active conjugates of bovine neurophysins and other polypeptides with multi-(poly-d,l-alanyl)-poly-l-lysine and their use to elicit antibodies

Fischer, Ernst; Curd, John G.; Chaiken, Irwin M.

doi:10.1016/0019-2791(77)90155-0

Cited by 21 publications

(11 citation statements)

References 23 publications

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“…Affi Prep 10 solid phase matrix and polystyrene cuvettes were from Bio-Rad Laboratories (Richmond, CA, USA). The antiRNase serum was obtained previously (Fischer et al, 1977). Affinity chromatographic elutions were operated with an FPLC LCC 500 system (Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability

Caliceti¹,

Schiavon²,

Veronese³

et al. 1990

J of Molecular Recognition

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The effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase-immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 x 10(-8) M was found for unmodified RNase, as compared to values of 1.3 x 10(-7) and 1.2 x 10(-6) M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine-2',3'-cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67 degrees C for native protein to 57 degrees C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability

Caliceti¹,

Schiavon²,

Veronese³

et al. 1990

J of Molecular Recognition

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“…The complete set of tryptic peptides was obtained by digestion after performic acid oxidation (Chaiken & Hough, 1980). The denotation of resultant peptides as "OT-number" follows the convention defined before (Wuu & Crumm, 1976;Chaiken 1 Abbreviations: NP-I and -II, bovine neurophysins I (oxytocin associated) and II (vasopressin associated); NP-I-(9-93) and [des-19,20]NP-I-(9-93), fragments derived from NP-I by trypsin digestion containing residues 9-93 and 9-18, 21-93, respectively; HPLC, highperformance liquid chromatography; TPCK-trypsin, L-l-(tosylamido)-2-phenylethyl chloromethyl ketone-trypsin; TEAP, triethylammonium phosphate buffer, prepared by adjusting 0.25 N phosphoric acid to pH 3.0 with distilled triethylamine; PBS, phosphate-buffered saline [the pH 7.4 solution was prepared as described previously (Fischer et al, 1977); the pH 7.3 solution was prepared from phosphosaline buffer (Calbiochem), pH 7.6 (containing 0.01% merthiolate), and titrated to pH 7. & Hough, 1980).…”

Section: Methodsmentioning

confidence: 99%

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Abercrombie¹,

Angal²,

Sequeira³

et al. 1982

Biochemistry

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Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.

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“…To do this, we have used previously prepared purified antibodies against neurophysin (15) (15). Other antibody preparations used were purified ribonuclease antibodies (15), antisera against oxytocin (Calbiochem), and nonimmune sera from rabbits prior to neurophysin immunization (15). Authentic bovine neurophysins I and II were isolated from posterior pituitaries essentially as described (15), with final purification by affinity chromatography on methionyltyrosylphenylalanyl-w-aminohexyl-agarose (18 (19).…”

mentioning

confidence: 99%

“…Other antibody preparations used were purified ribonuclease antibodies (15), antisera against oxytocin (Calbiochem), and nonimmune sera from rabbits prior to neurophysin immunization (15). Authentic bovine neurophysins I and II were isolated from posterior pituitaries essentially as described (15), with final purification by affinity chromatography on methionyltyrosylphenylalanyl-w-aminohexyl-agarose (18 (19). RNA was isolated from bovine hypothalami by using a modification (20) of the phenol extraction method of Aviv and Leder (21).…”

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confidence: 99%

See 1 more Smart Citation

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus

Giudice

Chaiken

1979

Proc. Natl. Acad. Sci. U.S.A.

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The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis.Poly(A-RNA was ' (of the translation product) were identical to major cysteinecontaining peptides of 'authentic neurophysin. The data show that hypothalamic mRNA directs the translation of'several unique cysteine-rich roteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteinecontaining tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

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Preparation of biologically active conjugates of bovine neurophysins and other polypeptides with multi-(poly-d,l-alanyl)-poly-l-lysine and their use to elicit antibodies

Cited by 21 publications

References 23 publications

Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability

Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus

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