In vitro cleavage specificity of the adenovirus type 2 proteinase

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.     

The comparison of seed protein patterns within the genusArachis by polyacrylamide gel electrophoresis

The seed protein patterns of 12Arachis species were compared by polyacrylamide gel electrophoresis (PAGE), similarities between patterns were measured by the Jaccard index. Results obtained confirm the close relationships established between members of the genus on morphological grounds and support the more recent classification schemes.A. villosa andA. correntina could well be regarded as distinct species on grounds of protein differences whileA. macedoi andA. villosulicarpa (although members of the same section, Extranervosae) show considerable differentiation of their protein patterns. Surprisingly, the formA. ×batizogaea showed less similarity in protein pattern to those of its parental species than might have been expected. The principle value of seed protein pattern data appears to be in distinguishing species within sections.     

Electro-optical measurements on aqueous suspension of purple membrane from Halobacterium halobium

The permanent dipole moment, polarizability, and the retinal angle of Halobacterium halobium purple membranes were determined at different pH values. All of the parameters have a maximum between pH 5 and 6. There is a reversal in the direction of the permanent dipole moment near pH 5. The value of permanent dipole moment was determined to be 60 D/protein at pH 6.6, and the value obtained for polarizability was 3 X 10(-28) Fm2/membrane fragment. The retinal angle of all-trans retinal was 0.8 degrees smaller than that of the 13-cis conformation.     

Reaction of organocuprate reagents with protected 1,2-anhydro sugars. Stereocontrolled synthesis of 2-deoxy-C-glycosyl compounds

The reaction of protected 1,2-anhydro-α-d-gluco- and β-d-manno-pyranoses with alkyl and phenyl organocuprates afforded the corresponding C-glycosyl compounds in acceptable to high yield. Complete stereocontrol was obtained, leading respectively to the β-d or the α-d anomer. With the perbenzylated manno derivative, deoxygenation at C-2 was achieved in high yield, affording 2-deoxy-α-d-C-glycosyl compounds.     

New C-nucleoside analogs by dehydration of 1-benzyl-4,5,6,7-tetrahydro-6,6-dimethyl-2-(d-galacto-pentitol-1-yl)-indol-4-one

The reaction between 2-(benzylamino)-2-deoxy-d-glycero-l-gluco-heptose and 5,5-dimethyl-1,3-cyclohexanedione yields 1-benzyl-4,5,6,7-tetrahydro-6,6-dimethyl-2-(d-galacto-pentitol-1-yl)-indol-4-one (2). Acid-catalyzed, intramolecular dehydration of 2 under kinetically controlled conditions gives 1-benzyl-4,5,6,7-tetrahydro-2-α-d-lyxofuranosyl-6,6-dimethylindol-4-one; the anomeric configuration of this compound is only suggested. When the dehydration reaction is conducted under thermodynamically controlled conditions, it produces a 1:1 mixture of the α- and β-d-lyxopyranosyl compounds. The structures of the new compounds were elucidated by chemical and physical methods.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Inhibition, by an amphiphilic substance, niflumic acid, of the inward rectification of the crustacean muscle fiber

Agents such as TEA+ or CS+ ions, these last ions instead of K+ ions in poor K extracellular solution, known to reduce or abolish the inwardly rectifying channel in many preparations produced no effect in crayfish muscle membrane By contrast, poor Cl extracellular solution (Cl- ions were replaced by CH3OSO3- ions) blocked the inward current activated by hyperpolarizing pulses and produced an increase of the resting potential. Niflumic acid is a agent which inhibited the inward going rectification of the crayfish muscle membrane. Apparent dissociation constant of niflumic acid with membrane sites was equal to about 6 X 10(-8) M; this value corresponds to that given by Cousin & Motais (1979) concerning translocation of Cl- ions in the membrane of red cells. Activation of the inward going rectification in the crayfish membrane is responsible of an inward current carried by Cl- ions.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

Determination of genotypes of human aldehyde dehydrogenase ALDH2 locus

Virtually all Caucasians have two major aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, in their livers, while approximately 50% of Japanese and other Orientals are "atypical" in that they have only ALDH1 and are missing ALDH2. We previously demonstrated the existence of an enzymatically inactive but immunologically cross-reactive material (CRM) in atypical Japanese livers. Among 10 Japanese livers examined, five had ALDH1 but not ALDH2 isozyme. These are considered to be homozygous atypical at the ALDH2 locus. Four had both ALDH1 and ALDH2 components detected by starch gel electrophoresis, that is, they are apparently usual. However, biochemical and immunological studies revealed that three of these four livers contained CRM. These three livers should be heterozygous atypical in the ALDH2 locus, that is, genotype ALDH2(1)/ALDH2(2). A Japanese liver, as well as control Caucasian livers, had no CRM, and they must be homozygous usual ALDH2(1)/ALDH2(1). Although the number of liver specimens examined is limited, the frequencies of three genotypes determined in this study are compatible with the values calculated based on the genetic model that two common alleles ALDH2(1) and ALDH2(2) for the same locus are codominantly expressed in Orientals. The remaining liver had only ALDH2 isozyme and was missing ALDH1. This type was not previously found in Caucasians and Orientals. The two-dimensional crossed immunoelectrophoresis revealed the existence of a CRM corresponding to ALDH1 in this liver. The abnormality can be considered to be due to structural mutation at the ALDH1 locus producing a defective ALDH1 molecule, although other possibilities such as post-translational modifications are not ruled out.