Robert I. Gregerman

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

Thyroxine-binding prealbumin (TBPA) in normal human serum has been shown in a polyacrylamide gel electrophoresis system to bind 7-9% of tracer level purified [(125)I]triiodothyronine (T3), and more than 30% of T3 in serum deficient in thyroxinebinding globulin (TBG). The T3-TBPA interaction has been confirmed at pH 9.0 and pH 7.4 in this electrophoretic demonstration of TBPA binding of T3 in serum. Purified human TBPA has also been shown to bind T3. Progressive additions of unlabeled thyroxine (T4) to serum containing tracer [(125)I]T3 displace T3 from TBG, its principal carrier, to TBPA and albumin; however, T4 loading does not lead to significant T3 displacement from TBPA even at T4 levels known to saturate TBPA. Loading of serum with unlabeled T3 results in displacement of more than 50% of [(125)I]T3 from TBPA, as well as from TBG, to albumin. Studies carried out with serum containing diphenylhydantoin (DPH) or MK-185, known inhibitors of T4 binding by TBG, also showed T3 displacement from TBG to TBPA and albumin. Although salicylate and tetraiodothyroacetic acid (TETRAC) displace T4 from sites on TBPA, they have only minimal effects on T3-TBPA interaction.

Epinephrine-and glucagon-sensitive adenylate cyclases of rat liver during aging. Evidence for membrane instability associated with increased enzymatic activity.

Abstract The influence of post-maturational aging on the activity and stability of rat liver epinephrine- and glucagon-sensitive adenylate cyclases (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) was studied in liver homogenates from male and female Wistar rats ages 3, 12, and 24 months. Enzyme activity was measured in the unstimulated (basal) state and with fluoride activation (5 mM) as well as with a range of concentrations of glucagon and epinephrine. Basal activity was the same at 3 and 12 months but at 24 months was 1.4 times higher in both sexes. With fluoride and glucagon (1 nM–10 μM) activities were slightly but significantly greater (1.2 times) at 24 months vs. 3 and 12 months in females but not in males. In contrast, epinephrine-stimulated activity doubled from 3 to 24 months in both males and females, with most of the increase occurring between 12 and 24 months. The concentration of epinephrine necessary for 50% activation was the same (0.25 μM) at all ages, showing that increased epinephrine response during aging is not due to altered affinity of receptors for hormone. Increased epinephrine-sensitive activity was due either to increased numbers of enzyme catalytic units or to an increase of V but not to altered K m for substrate which was unchanged with age for both hormone- and fluoride-activated enzyme. Stability of the membrane-bound enzyme was examined in homogenates kept at 0°C for 24 h in the presence of protease and esterase inhibitors. Loss of adenylate cyclase activity was significantly correlated with increasing age. At each age epinephrine activity was least stable, glucagon activity was most stable, and fluoride activity was intermediate. Glucagon-sensitive experimental deficiency of essential fatty acids, appears to correlate with decreased hormone responsiveness in vivo [42]. Moreover, during dietary manipulations altered fat cell adenylate cyclase activity is similarly associated with parallel changes of hormone-induced lipolysis [43]

Increased sensitivity to epinephrine stimulated lipolysis during starvation: tighter coupling of the adenylate cyclase complex.

Abstract Adipocytes of rats starved for 72 hr showed three-fold increase in sensitivity of the lipolytic response to epinephrine. Adenylate cyclase sensitivity in adipocyte membranes from starved rats was increased almost seven-fold over control rats. Binding studies using [ 3 H]-dihydroalprenolol showed that the number of β-adrenergic receptors per fat cell (1.1 × 10 5 and 1.1 × 10 5 receptors/cell, starved vs control) and the dissociation constant (5 vs 4 nM) of the receptor for the antagonist did not change following starvation. Increased sensitivity of adenylate cyclase activity in response to epinephrine without changes in the β-adrenergic binding parameters suggests that tighter coupling of adenylate cyclase with the receptor may mediate the more sensitive lipolytic response of starved rat adipocytes to epinephrine.

Parse Analysis: A New Method for the Evaluation of Investigators' Bibliographies

DURING the past five years our laboratory has been concerned with the development of a quantitative system for describing the relative contributions of authors to multiple-author manuscripts. The c...

Adenylyl cyclase activation by halide anions other than fluoride

Abstract Adenylyl cyclase of rat liver and fat cells is activated by chloride, bromide, and iodide in addition to fluoride, previously believed to be uniquely effective among the halide anions. Liver homogenates are activated approximately 6 fold by fluoride while chloride and bromide increase cyclase by 3 fold and iodide about 2 fold. Optimal concentrations of chloride, bromide and iodide are about 100 times higher than those required for activation by fluoride. The cyclase of fat cell ghosts is activated some 9 fold by fluoride, but the other halide anions produced effects very similar in magnitude to those seen with liver, although for fat the optimally effective concentrations were lower. These observations appear to relate adenylate cyclase to a number of other anion activated enzymes, some of which have already been studied in pure form by a number of physico-chemical techniques, and which may serve as models for understanding the action of fluoride and other anions on adenylyl cyclase.

Quantitation of beta adrenergic receptors in rat liver: confounding effect of displaceable but nonstereospecific antagonist binding.

In rat liver membranes three types of ligand binding were seen using [3H]-dihydroalprenolol (DHA) and [125I]-hydroxybenzylpindolol (HYP): binding stereospecifically displaced by beta-adrenergic agonists and antagonists, binding nonstereospecifically displaced by beta-adrenergic antagonists, and binding which was not displaced by beta-adrenergic agonists or antagonists. The magnitude of the nonstereospecific displaceable binding varied with the physiological state of the animal. It was sufficient to prevent the quantitation of the stereospecific displaceable binding in some preparations from young rats but in all preparations of rats greater than 150 g or more than about 6 weeks of age. In adrenalectomized weanling rats 10-30% of the total binding was of nonstereospecific displaceable type while in control rats it comprised up to 60% of the total binding. Addition of 5 X 10(-6) M phentolamine to the assay eliminated a large proportion of the nonstereospecific displaceable binding. When phentolamine was included in the assay, liver membranes from weanling rats stereospecifically bound 30-35% of total binding; membranes from adrenalectomized rats showed stereospecific binding of up to 50 to 80%. Because the amount of displaceable, nonstereospecific binding varied greatly depending on the physiologic state of the animals, stereospecific displacement should be monitored for every type of liver membrane preparation. Furthermore, animal age is an important variable. Using the published antagonist binding methodology (DHA or HYP) in liver membranes, it is not presently possible to quantitate liver beta-adrenergic receptors in normal rats that have reached maturity.

Human renin inhibition by a diazoacyl reagent: relationship of the enzyme to other proteinases.

Abstract Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N 2 CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renins inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.

(−) [125I] Iodopindolol Binding to β-Adrenergic Receptors in Tissues of the Rat with Particular Reference to the Characterization in Liver

The high affinity beta-adrenergic antagonist (-)[125I]iodopindolol was used to characterize and quantitate beta-adrenergic binding in liver and other tissues of the rat. Saturable, stereospecific binding with typical characteristics of beta-adrenergic receptors was demonstrated in all tissues examined (liver, lung, heart, kidney, fat and brain). Unlike the case with other radioactive ligands that have been used to measure beta-adrenergic receptors, assays were possible in crude homogenates as well as in purified membranes. Specific binding was defined as [125I]iodopindolol displaced by 0.1 mM isoproterenol. The percentage of [125I]iodopindolol specifically bound was greater than with other labelled antagonist ligands and it ranged from 95-60% of total binding over [125I]iodopindolol concentrations of 15-1000 pM. beta-Adrenergic binding could be quantitated in liver of animals of all ages whereas previously quantitation in liver was possible only using rats less than 2 months of age. Binding capacities ranged from a low of approximately 6 fmol/mg in liver particles precipitated at 4500 g to approximately 550 fmol/mg in 4500 g lung particles. The Kd of binding obtained by kinetic analysis was similar in all tissues. [125I]Iodopindolol is a nearly ideal ligand for the quantitation of beta-adrenergic receptors in various tissues of the rat. Because of its high affinity, stability and availability at high specific activity, this ligand should be especially useful in physiological studies requiring the accurate quantitation of receptor capacities.

Three Thyrotoxic Criminals

Abstract Three young men without previous criminal records developed thyrotoxicosis and committed felonies while symptomatically hyperthyroid. In each case the loss, or threatened loss, of gainful ...

Identification of histidylleucine and other histidyl peptides as normal constituents of human urine

Abstract In order to examine the possibility that the urinary excretion of histidylleucine might reflect the in vivo biogenesis of angiotensin, a technique has been devised for the detection of histidyl peptides in normal human urine. The peptides are isolated by displacement procedures on ion-exchange resins and further analyzed by paper and ion-exchange column chromatography. Histidyl peptides have been quantitated by fluorescence analysis after reaction with o-phthalaldehyde. Histidylleucine, in quantities of about 5–25 μg, has been shown to be present in 24-hour collections of human urine. The peptide isolated from urine has been found to be identical with synthetic material in comparisons using high resolution ion-exchange chromatography, paper chromatography in four solvent systems, acid hydrolysis followed by amino acid analysis, and fluorescence spectrum. A histidyl peptide which was regularly present, usually in somewhat larger quantities, was a tripeptide with sequence His-Leu-Gly or His-Gly-Leu. Numerous other histidyl peptides were present in smaller amounts but did not correspond to any of several other simple histidyl dipeptides examined and were not further identified.