A new method for determination of elastolytic activity using (14C) labeled elastin and its application to leukocytic elastase.

A new, highly sensitive and specific assay for elastolytic activity is described which employs insoluble elastin randomly labeled with [¹⁴C]. The substrate was prepared by labeling amino groups of the protein in vitro with [¹⁴C] methyl groups by reductive alkylation. The substrate was used to quantitate elastolytic activity from human leukocytes and to compare leukocytic elastase with pancreatic elastase. Purified human leukocytic elastase was approximately one-fourth as active as pancreatic elastase. Similar difference between leukocytic elastase and pancreatic elastase activities was found when the enzymes were tested against succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, but not when t-BOC-L-alanine-p-nitrophenyl ester was used.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

A trypsin-resistant heme peptide from cardiac cytochrome c1.

A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.     

Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane.

Band 3 is the predominant approximately 90,000-dalton polypeptide component of the human erythrocyte membrane. It was solubilized selectively, along with the other major glycoproteins, by extracting membrane ghosts with Triton X-100 under nondenaturing conditions. Two major polypeptides remained associated with Band 3 under these conditions; however one (Band 6) could be dissociated at an ionic strength of 0.15 and the other (Band 4.2) by treatment with p-chloromercuribenzoate. Band 3 was then purified (greater than or equal to 97%) by aminoethyl cellulose ion exchange chromatography. The isolated protein was free of phospholipid and was moderately enriched in apolar amino acid residues; it contained galactose and glucosamine but very little sialic acid and galactosamine. When Band 3 was labeled by treatment of ghosts with galactose oxidase plus KB3H4 and then purified, the electrophoretic mobility of its radioactivity lagged slightly behing that of its Coomassie blue staining profile. Variation in glycosylation could therefore cause the diffuse trailing zone characteristically observed for Band 3 on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The ultraviolet circular dichroism of Band 3 was stable in nonionic detergent and suggested an alpha helix content of 43%, a value close to that estimated for this polypeptide in the membrane.     

Interaction of flavins with electron-rich metals.

A complex of the electron-rich ion Cu(I) with the flavoquinone analogue 10-methylisoalloxazine has been synthesized and characterized by x-ray methods. The complex is unstable to oxygen. It is black-green in color, in contrast with the bright yellow, orange, or orange-brown crystalline complexes of 10-methylisoalloxazine or riboflavin with Cu(II), Ag(I), and Pb(II). These results are indicative of strong perturbation of the flavin electronic structure by the Cu(I) ion and suggest that this complex is a reasonable model for incipient transfer of an electron from a reduced metal to flavoquinone. the crystal structure is orthorhombic, Pna2-1, with unit cell constants a = 31.24(1) (figures in parentheses are estimated standard deviations), b = 12.862(4), c = 6.239(2) A, Pobs = 1.76 g per cm-3 and Pcalc = 1.77 g per cm-3 for Z = 4 and asymmetric formula CuClO4-2(C11H8N4O2). HCOOH. The final R factor based on 1250 counter-measured data is 8.8%. The 2 independent 10-methylisoalloxazine molecules, A and B, bind strongly to the cuprous ion throug N(5) of each flavin. The copper is approximately linearly coordinated with an N-Cu-N angle of 153(1) degrees, and Cu-N(5) distances of 1.94(2) A and 1.92(2) A. The next nearest atoms to Cu are the O(4) oxygens of each flavin, forming weak bonds with distances Cu-O(4) = 2.27(2) A and 2.21(2) A for molecules A and B. The dihedral angle between the 2 10-methylisoalloxazine molecules is 65.4 degrees.     

Nonsynaptic membrane of retinal horizontal cells as a slow potential amplifier

A simplified model of the membrane of horizontal cells of the L-type is designed to reflect two principal features of these cells previously studied experimentally: 1) their hyperpolarization response to light is the result of a decrease in the EPSP that is kept constant in darkness; 2) the resistance of their nonsynaptic membrane is reduced during hyperpolarization within physiological limits (from 0 to−70 mV). The model also reproduces properties of the horizontal cells such as the low membrane potential in darkness, reversal of the response to light during depolarization beyond the zero level, mutual amplification of color signals, saturation of the response to bright light, steady-state volt-ampere characteristics in darkness and light, and the amplitude characteristic curve which often has a steep part within a certain range of membrane potentials. The presence of hysteresis loops of the volt-ampere and amplitude characteristic curves of the horizontal cells predicted by the model was confirmed experimentally on the fish retina. Analysis of the model and results obtained with it show that the nonsynaptic membrane of the horizontal cells can actively amplify slow graded potentials.     

ZAKβ antagonizes and ameliorates the cardiac hypertrophic and apoptotic effects induced by ZAKα

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS‐dependent pathway and a mitochondria‐dependent pathway by subsequently inducing caspase‐3. ZAKβ, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKβ was demonstrated here using immunoprecipitation. The results show that ZAKβ has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKβ to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.