Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

Turnover of dihydrofolate reductase in rapidly dividing cells

The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [¹⁴C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2></mtext>}}$) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had $\text{t}{}_{\mathrm{<mtext>x1</mtext><mtext>2</mtext>}}$ values of approximately 3 and 9 hr.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

Stereoselective disposition of methadone in man.

Stable isotope labeled methadone (pentadeuteromethadone) has been used in conjunction with gas chromatography-chemical ionization mass spectrometry to study plasma disappearance rates and urinary excretion of pharmacologically active R-(?)-methadone (l-methadone) and inactive S-(+)-methadone (d-methadone) in three adult methadone maintenance patients. In all three cases, the analgesically active enantiomeric form of the drug had a significantly longer elimination half-life ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$) when studied in a steady state than did the inactive form ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$ for active R-(?)-methadone, 51.7 to 61.8 hours; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$ for inactive S-(+)-methadone, 31.8 to 37.0 hours). The ratio of drug elimination half-lives } R-(?)-/S-(+)- ranged between 1.40 and 1.94. In the two cases so studied, slower plasma disappearance of active R-(?)-enatiomer than the inactive S-(+)-enantiomer was also observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2\beta </mtext>}}$ R-(?)-, 42.8 and 52.5 hours; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2\beta </mtext>}}$ S-(+)-, 38.3 and 41.3 hours).     

Regulation of diamine oxidase expression by β2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the $\text{β}{}_2\text{-}\text{adrenergic}$ mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, $\text{β}{}_1\text{,β}{}_2\text{-}$ or $\text{β}{}_2\text{-}$, but not $\text{α}{}_1\text{-}$, $\text{α}{}_2\text{-}$, or $\text{β}{}_1\text{-}\text{receptor-blocking}$ this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine ($\text{α}{}_1$, $\text{α}{}_2$, $\text{β}{}_1$, $\text{β}{}_2$), isoproterenol ($\text{β}{}_1$, $\text{β}{}_2$) or terbutaline ($\text{β}{}_2$) showed that also in normal rat kidney diamine oxidase activity is under the control of $\text{catecholamine}\text{-β}{}_2\text{-}\text{receptors}$ through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered $\text{β}{}_2\text{-}\text{receptors}$ coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

Studies on the mechanism of activation of mitotic histone H1 kinase

A chromatin-associated histone H1 kinase has been detected in synchronized Novikoff hepatoma cells. Enzyme specific activity increased 4 to 6-fold from late G-2 to mid-metaphase, then decayed exponentially ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 28.5 min) to the interphase level. Extracts of the mitotic kinase retained the ability to decay $\text{in}\text{vitro}$ at 37°C but not at 0°C ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 24 min), under conditions in which interphase activity was stable. Sedimentation rates in sucrose density gradients of interphase and mitotic enzymes (before and after decay) were identical. Purification did not alter the rate of enzyme decay. However, high ionic strength prevented decay of crude but not purified preparations of mitotic enzyme. The results are discussed in terms of an allosteric mechanism for reversible activation of enzyme activity.     

The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, 1,3-dimyristoylglycerol-2-phosphoric acid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid.The phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 ($\text{p}\text{K}{}_1$). The regions between the first and the second $\text{p}\text{K}$ of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H⁺ concentration results in an abrupt change of the transition temperature. The slope of the $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagram beyond $\text{p}\text{K}{}_2$ becomes very steep. This is the     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.