In vivo formation and stability of engineered disulfide bonds in subtilisin

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.     

Thermodynamic parameters are sequence-dependent for the supercoil-induced B to Z transition in recombinant plasmids

The entropy and enthalpy changes which contribute to the thermodynamics of the B to Z transition were determined for three recombinant plasmids containing a (dC-dG)16 tract and for a plasmid containing a pair of (dT-dG)20 regions. For each base pair which adopts a left-handed conformation in the plasmids with (dC-dG)16 sequences, the delta HBZ and delta SBZ are -2.1 kcal/mol bp and -8.8 cal/K-mol bp, respectively. In the plasmid containing the (dT-dG)20 tracts, however, the delta HBZ and delta SBZ values are 0.58 kcal/mol bp and -0.76 cal/K-mol bp, respectively. Also, these determinations show that for each B-Z junction that forms in the plasmids containing the (dC-dG), the enthalpy and entropy changes are 24 kcal/mol junction and 65 cal/K-mol junction, whereas for the (dT-dG) plasmid, the enthalpy and entropy changes are -1.8 kcal/mol junction and -22 cal/K-mol junction, respectively. Those values for the enthalpy and entropy changes for the formation of a BZ junction in (dC-dG) and (dT-dG) plasmids suggest that the properties and possibly the structures of the junctions are different. Calculations using the enthalpy and entropy changes determined in this study reveal that the B to Z transition in plasmids containing (dC-dG) blocks are more temperature-dependent than the transitions in plasmids with (dT-dG) blocks. Surprisingly, at temperatures above 60 degrees C, calculations indicate that the B to Z transitions in (dT-dG) plasmids should be energetically favored over that transition in (dC-dG) plasmids.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

H2 histaminic receptors in rat cerebral cortex. 1. Binding of [3H]histamine

Saturable binding of [3H]histamine in equilibrium with homogenates of rat cerebral cortex reveals Hill coefficients between 0.4 and 1.0, depending upon the conditions. Data from individual experiments are well described assuming one or two classes of sites. Only the sites of higher affinity (KP1 = 3.9 +/- 0.5 nM) are observed when binding is measured by isotopic dilution at a low concentration of the radioligand (less than 1.5 nM) in the presence of magnesium or by varying the concentration of the radioligand. The sites of lower affinity (KP2 = 221 +/- 26 nM) appear during isotopic dilution at higher concentrations of the radioligand or at lower concentrations either upon the addition of guanylyl imidodiphosphate (GMP-PNP) or upon the removal of magnesium. Estimates of the second- and first-order rate constants for association and dissociation of [3H]histamine agree well with KP1. Apparent capacities corresponding to KP1 and KP2 are of the order of 100 ([R1]t) and 1300 pmol/g of protein ([R2]t), respectively. Simple interconversion cannot account for the changes in binding that occur upon adding GMP-PNP or removing magnesium, since the increase in [R2]t exceeds the decrease in [R1]t. Moreover, the apparent amount of high-affinity complex exhibits a biphasic dependence on the concentration of [3H]histamine; an increase at low concentrations is offset by a decrease that occurs at higher concentrations. The latter appears to be positively cooperative and concomitant with formation of the low-affinity complex. These and other observations indicate that the binding of histamine is inconsistent with models commonly invoked to rationalize the binding of agonists to neurohumoral receptors. GMP-PNP and magnesium reciprocally alter capacity at the sites of higher affinity, however, and the reduction caused by GMP-PNP reflects a substantial increase in the rate constant for dissociation at the sites that appear to be lost. The sites labeled by [3H]histamine thus reveal the properties of neurohumoral receptors linked to a nucleotide-specific G/F protein.     

H2 histaminic receptors in rat cerebral cortex. 3. Inhibition of [3H]histamine by H2 agonists

The binding of [3H]histamine to H2 receptors in homogenates of rat cerebral cortex is inhibited by 11 H2 agonists in a characteristic and unique manner. At low concentrations of the radioligand (less than 1.5 nM), the inhibitory profiles of individual agonists (A) are distinctly biphasic; specific binding is well described in most cases by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1. Maximal inhibition is the same for all agonists. Since values of F2 vary from 0.42 to 0.90, the agonist appears to determine the equilibrium distribution of receptors between two states of affinity. Ratios of apparent affinity (K2/K1) vary from 204 to 3 090 000, and there is no correlation between values of K1 and K2. Compounds lacking H2 activity, including structural analogues of histamine and dimaprit, reveal a Hill coefficient of 1 and inhibit the radioligand only weakly. For six agonists, values of K2 agree and correlate well (P = 0.00047) with H2 pharmacological potency (EC50) in the guinea pig right atrium; for the others, K2 is less than EC50 by 15-61-fold. Four observations suggest that the inhibition corresponding to F1 is allosteric and cooperative: the dissociation constant of the radioligand appears to vary in the presence of an unlabeled agonist, absolute levels of binding corresponding to F1, as defined by dimaprit, decrease at higher concentrations of [3H]histamine, F1 for dimaprit is reduced from 0.48 to 0.32 by 2-methylhistamine (F1 = 0.27) at a concentration of 20 nM (approximately K1(0.5) K2(0.5) for 2-methylhistamine), but the increase in K1 for dimprit is at least 100-fold less than expected from competitive effects, and 1 equiv of some agonists appears to preclude access of [3H]histamine to more than 1 equiv of receptors, with no evidence that an appreciable fraction of the unlabeled drug is bound. Noncompetitive effects also may account in part for the inhibition corresponding to F2.     

Fatty acid metabolism and ketone formation in the suckling rat

Rat milk triacylglycerols contain 35% medium-chain length fatty acids. About 70% of ingested medium-chain fatty acids are released from milk triacylglycerols in the stomach and small intestine and are absorbed directly into the portal venous system. Based on studies with the perfused suckling rat liver and in vivo studies with 2-tetradecylglycidic acid, an inhibitor of long-chain fatty acid oxidation, it is estimated that medium-chain fatty acids provide 75-80% of the substrate for ketogenesis. The preferential use of medium-chain fatty acids for ketogenesis spares long-chain fatty acids for complex lipid and membrane biosynthesis during this period of rapid growth. Although medium-chain fatty acids are the major substrate for ketogenesis, this pathway accounts for only 15% of the utilization of ingested medium-chain fatty acids, the rest presumably being oxidized directly in extrahepatic tissues.     

Detection of gonadotrophic hormones on isolated thecal interstitial cells of the domestic hen, Gallus domesticus, by an immunohistochemical method

Summary An immunohistochemical method was used to demonstrate the presence of gonadotrophins in isolated ovarian interstitial cells. The cells were obtained by collagenase digestion of large ovarian follicles after removal of the yolk and the granulosa layer. Using a peroxidase-labelled anti-rabbit serum with anti-chicken follicle stimulating hormone (FSH) serum raised in rabbits, a strong positive reaction was obtained. Anti-human FSH serum also produced a positive result but the reaction was weaker. There was no apparent difference in the staining reaction of cells which had been preincubated with ovine FSH serum. Treatment with anti-ovine luteinizing hormone (LH) resulted in a faintly positive reaction.The viability of the cells was tested by the Trypan Blue method and they were identified as steroid-producing cells by the histochemical demonstration of their 3-hydroxysteroid dehydrogenase activity.     

Chromosomal organization of chicken histone genes: preferred associations and inverted duplications.

We present a detailed picture of the disposition of core and H1 histone genes in the chicken genome. Forty-two genes were located within four nonoverlapping regions totalling approximately 175 kilobases and covered by three cosmid clones and a number of lambda clones. The genes for the tissue-specific H5 histone and other variant histones were not found in these regions. The longest continuous region mapped was 67 kilobases and contained 21 histone genes in five dissimilar clusters. No long-range repeat was evident, but there were preferred associations, such as H1 genes with paired, divergently transcribed H2A-H2B genes and H3-H4 associations. However, there were exceptions, and even when associations such as H1-H2A-H2B we maintained, the order of those genes within a cluster may not have been. Another feature was the presence of three (unrelated) clusters in which genes were symmetrically ordered around central H3 genes; in one such cluster, the boundaries of a duplicated H2A-H4 gene pair contained related repeat sequences. Despite the dispersed nature of chicken histone genes, the number of each type was approximately equal, being represented as follows: 6 H1, 10 H2A, 8 H2B, 10 H3, and 8 H4.