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S E Pattison

Publications and source records attributed to S E Pattison.

9 recordsLinked to original sources
PubMed1998

Zinc diffusion through lipid bilayers.

Zinc diffusion across liposome bilayers was measured for a set of phosphatidylcholines. These lipids were sonicated to form small unilamellar vesicles in the presence of the metallochromic indicator antipyrylazo III. This chelator sequentially forms two complexes with zinc ion. The rate constant for the first complex formation is shown to increase linearly with zinc concentration. The slope of this line, a [Zn2+]-independent, second-order rate constant, varies with changes in phosphatidylcholine properties. The rate constant is little affected by changes in fluidity as estimated from the reduced temperature [Tr = (Texperimental-Tc)/Tc]. In contrast, the rate constant is directly dependent on lipid oxidation as measured by either a thiobarbituric acid test or a spectrophotometric determination of conjugate dienes. We estimate that zinc diffusion stimulated by lipid oxidation can approach rates observed in hepatocyte zinc transport.

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PubMed1986

Kinetics of zinc uptake and exchange by primary cultures of rat hepatocytes.

The kinetics of 65Zn2+ uptake and exchange by hepatocytes in primary culture have been examined in detail to provide a basis for analyzing hormonal regulation of hepatic zinc metabolism. 65Zn2+ uptake was found to be a biphasic process. The slow phase represents an exchange between Zn2+ in the medium and preexisting, intracellular zinc pools. This exchange rate was saturable with a medium zinc concentration of 9.5 microM eliciting one-half the maximum exchange rate and a maximum exchange rate of 9.9 pmol Zn2+ . min-1 . mg protein-1 in the presence of bovine serum albumin. In the absence of albumin, a secondary, nonsaturable uptake rate was observed. The slow phase was relatively selective, and of the divalent transition metal ions tested, only Cd2+ and Mn2+ caused inhibition. The rate of exchange suggests total hepatocyte zinc has a turnover rate of approximately 30 h. The fast phase of 65Zn2+ reflects net Zn2+ accumulation into a labile pool. The initial rates for this process were too fast to be measured accurately, but steady-state measurements allowed determination of the labile pool size. The pool dimensions saturated in the presence [Kapp = 28.6 microM; pool capacity = 0.44 nmol Zn2+/mg protein] and absence [Kapp = 11.8 microM; pool capacity = 0.34 nmol Zn2+/mg protein] of bovine serum albumin. Kinetics and equilibria of Zn2+ uptake into the labile pool suggest that the latter acts as a source of Zn2+ for the slow-exchange phase. Dexamethasone stimulated slow Zn2+ exchange and also increased the labile pool size. The data suggest physiological factors alter hepatic zinc metabolism by influencing both intracellular Zn2+ pools.

Animals↗
PubMed1986

Zinc uptake and metabolism by hepatocytes.

Hepatocytes are in a dynamic equilibrium with the plasma zinc supply. Kinetic analysis of zinc uptake by isolated rat liver parenchymal cells defines two intracellular pools. In one pool zinc is bound relatively weakly and equilibrates rapidly with the medium at 37 degrees C. In the other pool zinc is bound tightly and interacts with the medium slowly at 37 degrees C. Of the two intracellular pools, the slower responding component represents an exchange process with the bulk of total cell zinc. The slow phase of uptake is saturable with albumin in the medium. The smaller pool is in rapid equilibrium with the medium and represents a labile zinc pool that accounts for net zinc accumulation. Both intracellular pools respond to hormonal stimuli. The factors that augment the uptake/exchange of zinc, namely glucocorticoids, glucagon, epinephrine, and dibutyryl cyclic AMP, are also those that stimulate metallothionein gene expression in hepatocytes. Changes in zinc flux into intracellular pools are directly related to the metallothionein content of hepatocytes. Characteristics of the labile zinc pool suggest that it may serve as an initial intermediate in zinc metabolism by hepatocytes as well as more general aspects of liver function related to zinc.

Animals↗
PubMed1976

On the mechanism of divalent metal ion chelator induced activation of the 7S nerve growth factor esteropeptidase. Activation by 2,2',2''-terpyridine and by 8-hydroxyquinoline 5-sulfonic acid.

Our previous studies (Pattison, S. E., and Dunn, M. F. (1975), Biochemistry 14, 2733) have shown that the reaction of divalent metal ion chelators with the 140 000 mol wt mouse submaxillary nerve growth factor protein (7S NGF) activates the iota-subunit esteropeptidase activity ca. sevenfold. Ultraviolet-visible spectral studies with the chelator 2,2',2''-terpyridine (terpyridine) and fluorescence emission studies with 8-hydroxyquinoline-5-sulfonic acid (HQSA) in combination with both conventional and rapid-mixing stopped-flow kinetic techniques have been employed in the present study to investigate (a) the mechanism of the chelator-induced activation process, and (b) the identity of the divalent metal ion involved. The spectral studies confirm the presence of stoichiometrically significant amounts of tightly bound zinc ion in native 7S NGF (1-2 g-atoms of An2+/mol of 7S NGF). The kinetic studies show that the reaction of terpyridine with 7S NGF occurs via a two-step process involving first a rapid, apparent second-order step (k1 = 1 x 10(6) M-1 s-1) to form a 7S NGF-Zn2+-chelator monocomplex, then a slow step to form a bis(terpyridine)-Zn(II) complex and activated 7S NGF in an apparent first-order process (kobsd = 0.10 min-1). This rate is, within experimental error, identical with the apparent first-order rate constant for the chelator-induced activation process (monitored by the rate of change in the steady-state rate of hydrolysis of chromophoric substrate, alpha-N-benzoyl-D,L-arginine-p-nitroanilide). Kinetic studies of the reaction of HQSA with native 7S NGF show that, under the same conditions of concentration, the rate of formation of the tris(HQSA)-Zn(II) complex is identical with the rate of the HQSA-induced activation of the 7S NGF esteropeptidase. Thus, these studies unambiguously establish that zinc ion is the metal ion involved in the chelator-induced activation process, and that activation involves removal of zinc ion from native 7S NGF.

Animals↗
PubMed1976

On the mechanism of divalent metal ion chelator induced activation of the 7S nerve growth factor esteropeptidase. Thermodynamics and kinetics of activation.

The 7S nerve growth factor protein (7S NGF) is a multisubunit zinc metalloprotein containing a masked trypsin-like esteropeptidase activity. Reaction of the native 7S NGF oligomer with divalent metal ion chelators effects an approximately sevenfold activation of the esteropeptidase activity via the sequestering and dissociation of the 7S NGF-bound zinc ion (Pattison, S. E., and Dunn, M. F. (1975), Biochemistry 14, 2733; Pattison, S. E., and Dunn, M. F. (1976), Biochemistry, preceding paper in this issue). In this study, investigation of the relationship between chelator concentration and the extent of activation, as measured by the steady-state rate of hydrolysis of alpha-N-benzoyl-D,L-arginine-p-nitroanilide, has demonstrated that (a) the chelator-induced activation is a freely reversible process, (b) activated 7S NGF undergoes a slow loss of reversibility when incubated with chelator over long time-periods, (c) the affinity constant of 7S NGF for zinc ion is approximately 10(10.5) +/- 10(0.5) M(-1), (d) chelator activation depends only on the ability of the chelator to sequester zinc ion, and (e) the activation process does not involve dissociation of the 7S oligomer to smaller subunit aggregates under conditions of low ionic strength.

Animals↗
PubMed1975

On the relationship of zinc ion to the structure and function of the 7S nerve growth factor protein.

The 7S nerve growth factor (7S NGF) is an oligomeric protein consisting of three distinct classes of subunits, alpha,beta, and gamma (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259). The beta subunit contains the growth promoting activity while gamma is a potent esteropeptidase. The proteolytic activity of gamma is virtually completely inhibited in the 7S NGF aggregate (L. A. Greene, E. M. Shooter, and S. Varon (1969), Biochemistry 8, 3735). In this paper, we report that divalent metal ion chelating agents effect a seven- to tenfold increase in the esteropeptidase activity of 7S NGF at pH 7.40. Plots of esteropeptidase activity vs. chelator concentration give saturation curves which are either sigmoidal (EDTA) or hyperbolic (o-phenanthroline) depending on the chemical structure of the chelator. A survey of common divalent metal ions shows that only zinc ion (Ki = 8 times 10(7) M) and, to a lesser extent, cadmium ion are effective, reversible inhibitors of both 7S NGF and the gamma subunit esteropeptidase activities. We have found that during isolation of 7S NGF, Zn2+ is selectively associated with the oligomer in a ratio of approximately 1-2 g-atoms of zinc/mol of 7S NGF with an apparent affinity which is orders of magnitude tighter than is indicated by the Ki value for the gamma subunit. Dialysis to pH 4.0 where 7S NGF is known to undergo a reversible dissociation (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259) brings about a tenfold reduction in the zinc ion content of the protein. This reduction is reversed on dialysis back to pH 7.4. In contrast, the isolated subunits contain only trace amounts of zinc ion at pH 7.4. Preliminary metal ion exchange experiments indicate that, of the common metal ions known to substitute for zinc in other zinc-metalloproteins, only cadmium ion is effective in substituting for zinc ion in 7S NGF. The fact that zinc ion is specifically bound to native 7S NGF, and that the zinc ion content of the system is critically dependent on the subunit aggregation state strongly suggests that zinc ion is an integral structural component of native 7S NGF.

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