James E. Zull

Interaction of the erythrocyte - membrane protein, spectrin, with model membrane systems

Abstract Spectrin causes an increased rate of solute diffusion from liposomes. The protein binds both to negatively charged and positively charged liposomes. Binding to negative liposomes appears to be insensitive to ionic strength of the medium. It appears that hydrophobic bonding of this membrane protein to lipid is the likely mode of interaction in the erythrocyte membrane.

Is genetic code redundancy related to retention of structural information in both DNA strands

We have noted that the sense-antisense relationships inherent in the genetic code divide the amino acids into three separate groups. The nature of the amino acids in each group may allow the polypeptides coded by the antisense strand to retain the secondary structure patterns of the translated strand. Also, this relationship requires all but eight of the codons in the eukaryotic code and all but four in the mitochondrial code. Thus, genetic code redundancy could be related to evolutionary pressure toward retention of protein structural information in both strands of DNA.

Activation of glucose diffusion from egg lecithin liquid crystals by serum albumin.

Abstract The diffusion of [ 14 C]glucose from phospholipid model membrane systems has been measured in the presence and absence of bovine serum albumin under various conditions. At acid pH, bovine serum albumin enhances the rate at which glucose diffuses from lecithin-cholesterol-dicetyl phosphate micelles. The enhancement effect has a p K of 4.3. This is close to the pH at which the well-known acid conformation change occurs in this protein. The activation of diffusion only takes place with negatively charged micelles, and is not disrupted by high salt concentrations. The effects of temperature and protein concentration on the diffusion rate were also studied. It is suggested that the protein binds through electrostatic interactions initially, but that the subsequent formation of apolar bonds brings about the activation of glucose diffusion.

The binding of serum albumin to phospholipid liposomes

Abstract The binding of bovine serum albumin to phospholipid model membrane systems has been studied as a function of pH, ionic strength and membrane charge. At pH values below its isoelectric point, bovine serum albumin becomes strongly bound to lecithin-cholesterol-dicetyl phosphate liposomes. The dependence of the binding on ionic strength and membrane charge indicates that actually two types of association are possible: electrostatic and hydrophobic binding. The electrostatic interactions seem to be a prior condition for the formation of the apolar associations which are related to the acid expansion of bovine serum albumin occurring at pH 4.0. It is suggested that the formation of an apolar complex mediated through an initial electrostatic attraction gives rise to the changes in permeability properties of the model membrane which were studied previously.

The role of the methionine residues in the structure and function of parathyroid hormone

Forms of the biologically active N-terminal fragment of bovine parathyroid hormone oxidized at methionine 8, methionine 18, and both positions were prepared, separated from one another, and characterized as described earlier for the native hormone (A. L. Frelinger and J. E. Zull, (1984) J. Biol. Chem. 259, 5507). The biological properties of the oxidized forms were compared to those of the native hormone, using the renal membrane adenylyl cyclase assay. Oxidation at position 18 produced full agonists of the hormone with slightly reduced potency. Oxidation at position 8 produced partial agonists of greatly reduced potency. Oxidation at both positions produced partial agonists of even lower potency. Thus, methionine 8 is implicated both in binding and in activation of adenylyl cyclase, but methionine 18 is implicated only in binding. Further study showed that oxidation of both residues is dependent on the pH, ionic strength, and polarity of the solvent. However, methionine 8 is less easily oxidized than methionine 18. This difference is eliminated in 3 M guanidine-HCl with 1-34 and in 6 M guanidine-HCl with 1-84. On the other hand the difference in reactivity is greatly increased in high ionic strength, with methionine 8 becoming much less reactive. These results suggest that the methionine residues are important in the biologically active conformation of parathyroid hormone and that methionine 8 is less accessible than methionine 18 under certain conditions. These conclusions are discussed in the context of a specific model for the folding of parathyroid hormone.

Problems and approaches in covalent attachment of peptides and proteins to inorganic surfaces for biosensor applications.

SummarySome of the fundamental problems in covalent attachment of peptides and proteins to putative biosensor surfaces are reviewed and specific approaches to these problems discussed. In addition, selected aspects of our recent work utilizing self-assembled monolayer (SAM) systems designed to react selectively with the thiol side chain of Cys in proteins are presented. Uniform attachment of a 21-amino acid peptide antigen through a single Cys residue with retention of biological function (antibody binding) has been attained. Further work with this system may lead to solutions for some of the problems which currently prevent the development of reliable biosensors for industrial and medical use.

Interactions of parathyroid hormone and plasma membranes from rat kidney

Summary The initial interaction between parathyroid hormone and isolated purified membrane preparations from rat kidney is probed with the use of a highly tritiated, biologically active derivative of parathyroid hormone, [ 3 H] acetamidino-parathyroid hormone. A method for assaying the binding of the labeled parathyroid hormone to these membranes at nanogram per milliliter concentrations has been demonstrated utilizing this method. The binding described is not reduced by pre-incubations with large excess quantities of other polypeptide hormones, but is qualitatively abolished by pre-incubation with smaller quantities of unlabeled parathyroid hormone. The oxidized, biologically inactive labeled parathyroid hormone does not bind to these membranes.

Proton NMR studies of the biologically active 1–34 fragment of bovine parathyroid hormone: Examination of a structural model

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.3. Denatured PTH showed only one histidine C-2 peak with a pKa of 6.7. An aliphatic peak identified as due to either a methionine or a glutamine residue also shifted with pH, and the pKa for this shift was 6.3. Finally, small but significant upfield shifts in the methyl and methylene resonances were observed as a function of pH, and when compared to the denatured peptide. These results indicate that the N-terminal domain of native PTH has considerable structure in solution, and are consistent with a theoretical model for the folding of this peptide.

Interaction of kidney (Na+−K+)-ATPase with phospholipid model membrane systems

The transport-related, Na+, K+-activated ATPase from kidney can be solubilized by treatment with the non-ionic detergent, Lubrol. The Lubrol-solubilized enzyme does not require phosphatidylserine for activation. This enzyme preparation binds to positively charged, but not to negatively charged, liposomes. It also binds to sphingomyelin liposomes which are formally neutral in charge, but not to lecithin. The bound enzyme can be extracted with solutions of high ionic strength.

The development of hormonal responses of cultured embryonic chick limb mesenchymal cells

Abstract Cells obtained from stage 24 chick limb buds were cultured and assayed for their ability to respond to exogenously supplied parathyroid hormone (PTH) as monitored by analysis of cellular cyclic AMP (cAMP). After 3–4 days in culture, these cells developed a striking responsiveness to the hormone; 20 -to 50-fold elevations in cAMP were routinely observed upon exposure to 10−8, M hormone for 2 min. This response was greatest in cells initially plated at low densities (1 × 106 cells/35-mm dish) and was inversely correlated to the amount of cartilage which developed in such cultures. Cells obtained from limbs of stages 23–26 embryos developed a similar responsiveness to PTH after 3–4 days in culture, but cells obtained from limbs of stage 22 embryos showed no such responsiveness even after 6 days in culture. A response to calcitonin also was noted in cultures of stage 24 limb mesenchymal cells after 4–5 days in culture, but this was of much smaller magnitude than the PTH response. Of 12 other hormones tested, only β agonists elicited any cAMP response in the cultered stage 24 limb mesenchymal cells. Although cells initially plated at a high density and grown for 8 days in culture show no response to PTH, the presence of PTH-responsive cells in such cultures could be demonstrated by sequential digestion with collagenase and replating the extracellular matrix-free cells released by this treatment. Such replated cells then exhibited a responsiveness to PTH. Thus, the responsiveness of cultured limb mesenchymal cells depends on the developmental stage of the starting limb mesenchyme, the phenotypes which develop, and physical factors such as accessibility to exogenously supplied hormone.