PubMed Health⌕ Search

Biomedical subjects

R E Shepherd

Publications and source records attributed to R E Shepherd.

At least 55 records · Page 3Linked to original sources
PubMed1975

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.

Adenosine Deaminase↗
PubMedDate not supplied

Comparison of energy-minimized structures of [Pd(II)(N-methyliminodiacetate)] complexes of X(1)-His-X(3)-His-His peptides as an analysis of steric and specific interactions with synthetic binding tags for IMAC separations.

[Pd(II)(mida)(peptide)] complexes for the series of peptides of sequence X(1)-His-X(3)-His-His were studied by molecular mechanics methods using Spartan, MMFF94, and SYBYL programs with X(1) = X(3) = glycine (G), phenylalanine (F), tyrosine (Y), tryptophan (W), and with X(1) = glycine (G) and X(3) = proline (P). For comparison purposes, data were also obtained for the Ser-Pro-His-His-Gly (SPHHG) and the (His)(5) peptides.The latter two peptides and GHPHH are tags in current use for IMAC separations. These provide calibration points as to the binding affinities that have been determined for the entire series. The energies of the complexes, as an average trend found from the composite behavior of the three methods, were found to be SPHHG (205 kcal/mol) (most stable; are values obtained by MMFF94 methods) < HH(#)HH(#)H(#) (222; where # implies the site of attachment to match the other X(1)-His-X(3)-His-His peptides) < YHYHH (249) < GHGHH (265) < WHWHH (284) approximately GHPHH (286) < FHFHH (311) (least stable), implying that FHFHH might be a useful chromatographic tag for IMAC protein separations that would elute more readily than GHPHH from IMAC sites that are of square-planar structure, such as Cu(II)(ida-supported) IMAC columns. Specific H-bonded interactions are observed between the tyrosine X(1) and pendant carboxylates and between X(3) and the N-terminal amine of [Pd(mida)(YHYHH)]. Face-to-pi-face ring stacking occurs between phenylalanine X(1) and X(3) units in [Pd(mida)(FHFHH)], whereas edge C-H to pi H-bonding or pi stacking occurs between the X(1) and X(3) tryptophans of [Pd(mida)(WHWHH)]. Two energy minima were found with tryptophan. The more stable form has the aromatic rings more parallel, similar to the stacked form of phenylalanine, rather than the edge C-H to pi H-bonding, and virtually the same overall energy as for [Pd(mida)(GHPHH)]. The "perpendicular" structure was found as an initial local energy minimum, but additional MMFF94 calculations found the pi -stacked arrangement at energy ca. 39 kcal/mol lower than that of the nearly "perpendicular" arrangement of the tryptophan rings, a composite effect of relaxation of the peptide, together with differences in stabilities imparted by the differing geometries. The use of the terms "pi-stacked " and "perpendicular" forms represent the limiting cases available to the tryptophan side chain groups. A twist of about 15 degrees to 20 degrees in dihedral angle is all that is necessary to change between structures that are nearly described as one form or the other.

Chromatography, Affinity↗
PubMedDate not supplied

Design of affinity tags for one-step protein purification from immobilized zinc columns.

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Affinity Labels↗