摘要:

AbstractThis paper describes the enzyme catalyzed synthesis of the undecapeptide substance P from its non‐associated fragments (1–7) and (8–11) or (1–8) and (9–11). The fragment condensation was mediated by the use of product specific antibodies as molecular traps. As catalyst a previously purified endopeptidase was used which specifically hydrolyzes substance P at the Phe7–Phe8and Phe8–Gly9bonds. The synthesis was performed in analytical scale and product formation was guided by reversed phase HPLC combined with radioimmunoassay. It appeared that the substance P fragments (1–8) and (9–11) were condensed to a larger extent than (1–7) and (8–11). This observation may well result from the higher affinity of the antibodies observed for substance P (8–11) as compared to that found for the other fragments. Increased concentration of the antibodies also seemed to result in enhanced res

ISSN:0952-3499    

DOI:10.1002/jmr.300010202

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractExact equations which describe the kinetic patterns of enzyme/enzyme complexes, when compartmented coupling occurs between them, are presented. Compartmented coupling refers to the creation of a local environment in which the concentration of an intermediate, shared by two enzymes, is higher than its solution concentration. This result in a higher coupling enzyme activity, a condition reflected in a shorter transition time for the system. In this paper, equations are presented which allow experimenters to quantitate the effect of compartmented coupling in terms of changes in the apparentKmandVmaxvalues. The equations presented in this paper are more exact than those previously derived since they do not incorporate first order assumptions before derivation.

ISSN:0952-3499    

DOI:10.1002/jmr.300010203

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractStreptococcal protein G is an IgG‐binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG‐binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG‐ and albumin‐binding, different parts of the receptor molecule were produced inE. coliusing a coupled expression/secretion system. Affinity Chromatography, using IgG or HSA immobilized on Sepharose, Showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin‐binding. The structure of this part of the proteins suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one‐step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification i

ISSN:0952-3499    

DOI:10.1002/jmr.300010204

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractThe synthesis of an 11 membered ring bis‐lactam, a system which is designed as a conformationally restricted mimetic of type I and II β‐turns is described. Computer assisted molecular modeling was used to compare the predicted low energy conformers of the turn mimetic with idealized type I and type II turn structures. Initial computation analysis indicates that the basic ring structure will provide an excellent foundation for the development of variety of β‐turn m

ISSN:0952-3499    

DOI:10.1002/jmr.300010205

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractWe have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+and Ni2+ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3Murea‐induced transformation to the DNA‐binding form, and (2) limited trypsin digestion to separate the steroid‐ and DNA‐binding domains. Imidazole (2–200 mM) affinity elution and pH‐dependent (pH 7–3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+and Ni2+ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+>Cu2+» Zn2+) at neutral pH. Generally, similar results were obtained with IDA–Cu2+and IDA–Ni2+in the absence of urea. Receptors were tightly bound and not eluted before 100 mMimidazole or pH 3.6. Different results were obtained using IDA–Zn2+; at least four receptor isoforms were resolved on IDA–Zn2+. Receptor–metal interaction heterogeneity and affinity for IDA–Zn2+and IDA–Cu2+, but not IDA–Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA–Zn2+chromatography were not separable using high‐performance size‐exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA‐affinity chromatography. The affinity of trypsin‐generated (mero) receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High‐affinity metal‐binding sites were mapped to the DNA‐binding domain, but at least one of the metal‐binding sites is located on the steroidbinding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor‐metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal‐binding proteins suggests new and potentially powerful receptor immobilization and purification

ISSN:0952-3499    

DOI:10.1002/jmr.300010206

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractAnhydroelastase was effectively isolated by a single operation of affinity chromatography from a complex mixture produced by phenylmethylsulfonylation and alkaline treatment of porcine pancreatic elastase. The adsorbent used for the chromatography was 6‐aminohexanoyl‐trialanine, which corresponds to a product of elastase action, immobilized on Sepharose 4B. Successful resolution by the operation indicated that this immobilized ligand possesses the highest affinity for anhydroelastase among various proteins including regenerated elastase in the mixture. Comparative affinity chromatography on immobilized anhydroelastase and on immobilized native elastase further confirmed the stronger interaction of anhydroelastase with the product‐type peptides. Immobilized anhydroelastase was also found to be useful in the purification and search for naturally occurring proteinase inhib

ISSN:0952-3499    

DOI:10.1002/jmr.300010207

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

摘要:

AbstractMost antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the α‐helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I‐Ad. Hybridoma A.1.1 responds to EYK(EYA)4as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the α‐helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC mo

ISSN:0952-3499    

DOI:10.1002/jmr.300010208

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY

ISSN:0952-3499    

DOI:10.1002/jmr.300010209

出版商:John Wiley&Sons, Ltd.    

年代:1988

数据来源: WILEY