The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.     

The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor

Bacitracin was found to be an effective inhibitor of the $\text{in}\text{vitro}$ degradation of both thyrotropin releasing factor¹ (TRF) and luteinizing hormone releasing factor (LRF) by guinea pig hypothalamic and whole brain homegenates and rat hypothalamic homogenates and subcellular fractions. Bacitracin was effective in inhibiting the degradation of TRF and LRF, as determined by radioimmunoassay, where it exhibited no interference with the assays. Kinetic studies of the degradation of exogenous synthetic [³H]-TRF demonstrated non-competitive inhibition by bacitracin with K_i = 1.9 × 10^?5 M, while studies on the degradation of [³H] LRF indicated competitive inhibition with K_i = 1.7 × 10^?5 M. Electrophoretic and amino acid analysis revealed that bacitracin itself was not degraded during the course of the $\text{in}\text{vitro}$ incubation.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

Bioreduction of nitroxides by Staphylococcus aureus.

Two nitroxide radicals (TEMPO, I; OXAN, II) and a spin labeled penicillin (III) were reduced by $\text{Staphylococcus aureus}$. A short induction period preceded zero order reduction of these substrates leading to a K_m of 8 × 10^?4M, 6.67 × 10^?5M and 5.7 × 10^?4M and V_max of 106, 26 and 11 μ mole/min mg bacteria for I, II and III, respectively.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Identification of a structural hairpin in the filamentous chimeric phage M13Gori1

Crystals of alkaline phosphatase (EC 3.1.3.1; M_r 94,000) grown at pH 9.5 from 2.25 m-(NH₄)₂SO₄ with 5 × 10^?5 m-Zn and 10^?2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH₄)₂SO₄ with 10^?2 m-Zn and 10^?2 m-Mg present. The crystals are orthorhombic with $\text{a = 195.5}\text{A}\text{?}\text{, b = 168.3}\text{A}\text{?}\text{and}\text{c = 76.33}\text{A}\text{?}$, and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K₂PtCl₄, KAu(CN)₂ and K₂OsO₄ derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major $\text{α}\text{β}$ domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the $\text{α}\text{β}$ domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the $\text{α}\text{β}$ domain. In both there is extensive monomer-monomer contact.Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10^?2 m or cadmium at 10^?2 or 5 × 10^?2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10^?2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate.The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10^?3 m, 1.5 × 10^?1 m and 1.3 × 10^?2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α and PGF_2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K_s) were highest with PGA₁ and PGA₂. With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a $\text{weak}$ spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA₁ and PGA₂ yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA₁ and PGA₂ to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA₁ was competitive; the K_i = 8.2 × 10^?4 M was found to be similar in magnitude to the K_s = 7.3 × 10^?4 M of PGA₁ observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.     

Practical acid dissociation constants,temperature coefficients,and buffer capacities for some biological buffers in solutions containing dimethyl sulfoxide between 25 and −12 °C

Acid dissociation constants (K_a), usually expressed as pK_a (-logK_a) can be considered as indices of acid-base equilibria in solution and their evaluation under the solution conditions that exist during the exposure of biological systems to low temperatures are as important as the measurement of pH per se. The assignment of pH1 standards to define pH1 scales in the binary mixed solvent, dimethyl sulfoxide-water (27), has provided the basis for measuring the pK_a1 values of some biological buffers in mixtures of Me₂SO and H₂O which have particular relevance to studies which demonstrate the “pH-dependent” recovery of smooth muscle after low-temperature storage (9, 31). “Practical” ionization conslants in water (pK′_a) and in 20% ($\text{w}\text{w}$) and 30% ($\text{w}\text{w}$) dimethylsulfoxide-water (pK_a1) have been measured by potentiometric titration of a range of zwitterionic buffer compounds at 25, 0, ?5.5, and ?12 °C together with the respective buffer capacities and temperature coefficients. Measurements have been made with reference to the relevant standard states for each solvent system, thereby endowing the values with as much thermodynamic significance as possible.     

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10¹⁰ sites per 100 μg protein in the youngest group (Pool A) to 46 × 10¹⁰ sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ (“empty site”), was 1.53 × 10⁸M^?1 in Pool A and was only slightly higher than $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ (“filled site”). $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ was higher in Pool B, 1.75 × 10⁸M^?1, and in Pool C reached a value of 5.63 × 10⁸M^?1. In Pool C $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ was 2.3 × 10⁸M^?1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10¹⁰ sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10¹⁰). In addition, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ increased from 0.75 × 10⁸M^?1 at 14 days to 3.02 × 10⁸M^?1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.     

Photoaffinity labelling by S-(p-azidophenacyl)-glutathione of glyoxalase II and glutathione S-transferase

S-(p-azidophenacyl)-glutathione, $\text{l}$, is a linear competitive inhibitor at pH 7.40 of beef liver glyoxalase II with K_i = 7.96 × 10^?4 M. On irradiation at 340 nm it covalently inhibits glyoxalase II to a level of 42 ± 5% inhibition. This photoaffinity labelling is prevented by the presence of a glyoxalase II competitive inhibitor (the hemimercaptal of glutathione and methylglyoxal). A crude preparation of sheep liver glutathione S-transferases is also irreversibly inactivated (86% ± 5% inhibition) by irradiation at 320 nm in the presence of $\text{l}$.     

A "Reverse burst" active site titration procedure for human carbonic anhydrase B.

Between pH 7 and pH 10.5, p-nitrophenyl $\text{p}$-sulfamyl benzoate (PNP-SAB) binds very strongly to human carbonic anhydrase B (dissociation constant on the order of 10^?9 M or less at pH 7.5), but is not hydrolyzed by the enzyme. Because the binding is essentially stoichiometric under readily accessible conditions, this ester may be used as an active site titrant, by measuring the rapid hydrolysis of excess unbound PNP-SAB catalyzed by an added nucleophile (“reverse burst”).