The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Acid secretion and the H,K ATPase of stomach.

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.     

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.     

Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.     

Partitioning of fluorescent phospholipid probes between different bilayer environments. Estimation of the free energy of interlipid hydrogen bonding.

Fluorescence spectroscopy has been used to monitor the partitioning of a series of exchangeable neutral phospholipid probes, labeled with carbazole, indolyl or diphenylhexatrienyl moieties, between large unilamellar vesicles containing 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) or N-hexadecyl-N-(9-octadecenyl)-N,N-dimethylammonium chloride (HODMA). Phosphatidylethanolamine (PE) probes desorb from POPC-containing vesicles at markedly slower rates than do phosphatidylcholine (PC) probes with the same acyl chains. The rate of probe desorption from such vesicles is progressively enhanced by successive N-methylations of the amino group but not by methylation of C-2 of the ethanolamine moiety, a modification that leaves unaltered the hydrogen-bonding capacity of the polar headgroup. By contrast, the rates of desorption of different probes (with the same acyl chains) from HODMA or from DOTAP vesicles are much more comparable and reflect no clear systematic influence of the headgroup hydrogen-bonding capacity. Equilibrium-partitioning measurements indicate that the relative affinities of different probes for PC-rich vesicles, in competition with HODMA or DOTAP vesicles, increase with increasing hydrogen-bonding capacity of the probe headgroup in the order PC less than N,N-dimethyl PE less than N-methyl PE less than PE approximately phosphatidyl-2-amino-1-propanol. From such partitioning data, we estimate that interlipid hydrogen-bonding interactions (in competition with lipid-water interactions) contribute roughly -300 cal mol-1 to the free energy of a PE molecule in a hydrated liquid-crystalline phospholipid bilayer; this free-energy contribution is somewhat smaller, but still significant, for N-mono- and dimethylated PE's.     

Microscopic versus macroscopic diffusion in model membranes by electron spin resonance spectral-spatial imaging.

The macroscopic and the microscopic diffusion coefficients of a phospholipid spin label (16-PC) in the model membrane 1-palmitoyl-2-oleoyl-sn-glycero-phosphatidylcholine have been measured simultaneously in the same sample utilizing the new technique of spectral-spatial electron spin resonance imaging. The macroscopic diffusion coefficient Dmacro for self-diffusion of 16-PC spin label is obtained from imaging the concentration profiles as a function of time, and it is (2.3 +/- 0.4) x 10(-8) cm2/s at 22 degrees C. The microscopic diffusion coefficient Dmicro for relative diffusion of the spin probes is obtained from the variation of the spectral line broadening with spin label concentration, which is due to spin-spin interactions. Dmicro is found to be substantially greater than Dmacro for the same sample at the same conditions, and is estimated to be at least (1.0 +/- 0.4) x 10(-7) cm2/s. Possible sources for their difference are briefly discussed in terms of the models used for Dmicro.