Febs Letters

Demchenko, Alexander P.

doi: 10.1016/0014-5793(92)81334-Ipmid: 1327871

Biocatalytic reactions can occur according to two very different mechanisms: homogeneous, which is described by standard transition state theory (TST) and its modifications, and inhomogeneous (polychromatic), which is characteristic for some of the charge‐transfer reactions in liquids and amorphous solids. While most data published on enzyme reactions are interpreted on the basis of homogeneous kinetics, the important recent findings suggest the involvement of inhomogeneous kinetics mechanism.

Matsui, Ikuo; Ishikawa, Kazuhiko; Miyairi, Sachio; Fukui, Sakuzo; Honda, Koichi

doi: 10.1016/0014-5793(92)81335-Jpmid: 1397275

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera α‐amylase was altered to arginine (R) or asparagine (N) by site‐directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three‐times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.

Lew, Roger R.; Garrill, Ashley; Covic, Lidija; Heath, I.Brent; Serlin, Bruce S.

doi: 10.1016/0014-5793(92)81336-Kpmid: 1397276

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K+ ion channels (2–6 pA) using the patch‐clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double‐barreled micropipettes; thus, inward K+ flux is due solely to concentration differences. Although conductances are in the range expected for K+ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch‐clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.

Moreau, Patrick; Juguelin, Hélène; Cassagne, Claude; Morré, D.James

doi: 10.1016/0014-5793(92)81337-Lpmid: 1397277

The molecular basis for temperature compartment formation was investigated using a cell‐free system from rat liver. The donor was from liver slices prelabeled with [3H]acetate. Unlabeled Golgi apparatus membranes were immobilized on nitrocellulose as the acceptor. When transfer was determined as a function of temperature, a transition in transfer activity was observed at low temperatures (≤ 20°C) similar to that seen in vivo. The decrease in transfer efficiency correlated with a decrease in phosphatidylethanolamine and phosphatidylserine content of the transition vesicles formed. By adding lipid mixtures enriched in these lipids to the vesicles, their ability to fuse with the cis Golgi apparatus was reconstituted. These findings provide evidence for a role for lipids in low temperature compartment formation.

Strauss, John D.; Zeugner, Claudia; Van Eyk, Jennifer E.; Bletz, Christel; Troschka, Monika; Rüegg, J.Caspar

doi: 10.1016/0014-5793(92)81338-Mpmid: 1397278

Calcium‐dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate‐free, low‐Ca2+ solution (pCa > 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca22+ (pCa ∼4.5 prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% ± 18.7% of control force). SDS‐PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.

Seidah, N.G.; Fournier, Hélèrie; Boileau, Guy; Benjannet, Suzanne; Rondeau, Nomand; Chrétien, Michel

doi: 10.1016/0014-5793(92)81339-Npmid: 1397279

The complete cDNA structure of the porcine (p) pro‐protein and pro‐hormone convertase PC2 (pPC2) was obtained from a cDNA library of pituitary neurointermediate lobes mRNA. The deduced amino acid sequence revealed that pPC2 exhibits a 99‐97% sequence identity to the human, mouse and rat homologues. The 3′ end of the 2.1 kb cDNA is the least conserved segment. On Northern blots of pars intermedia poly A+ RNA two transcripts of 3 and 5 kb were detected. Molecular analysis of the N‐terminal glycopeptide products of porcine pro‐opiomelanocortin (pPOMC) co‐expressed with vaccinia virus recombinants of PC1 or PC2, revealed that in cells devoid or containing secretary granules both convertases can cleave pPOMC with PC1 releasing the 1–80, 1–107 and 1–148 glycopeptide fragments, and PC2 cleaving pPOMC directly into pPOMC 1–107.

Rogers, W.John; Hodges, Michael; Decottignies, Paulette; Schmitter, Jean-Marie; Gadal, Pierre; Jacquot, Jean-Pierre

doi: 10.1016/0014-5793(92)81340-Rpmid: 1327872

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N‐ and C‐termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr‐7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.

Miyamoto, Kazumasa; Nishimura, Koichi; Masuda, Tatsuru; Tsuji, Hideo; Inokuchi, Hachiro

doi: 10.1016/0014-5793(92)81341-Ipmid: 1397280

The accumulation of protoporphyrin IX (Proto IX) in light‐sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild‐type strain CA274 or in visA mutants that had been rendered light‐resistant by introduction of the wild‐type visA + gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.

Borrello, Silvia; De Leo, Maria Emilia; Wohlrab, Hartmut; Galeotti, Tommaso

doi: 10.1016/0014-5793(92)81342-Jpmid: 1397281

The presumed involvement of the transition metals manganese and copper in the regulation of the expression of the Mn‐ and CuZn‐containing superoxide dismutase genes has been investigated in normal and neoplastic tissues of the rat. Two hepatomas of the Morris line have been employed, the slow growing, highly differentiated 9618A and the fast growing, poorly differentiated 3924A. The data obtained indicate a control at the pretranslational level of the Mn‐containing enzyme, presumably exerted by the manganese ion. The CuZn‐containing superoxide dismutase is also regulated pretranslationally in the normal tissues examined and in the hepatoma 3924A. However, there is no indication for the involvement of the copper ion, which in the liver is mostly located in the cytosol bound to CuZnSOD, in such regulation. The possible role of a reduced redox state, concomitant to the manganese deficiency in hepatoma tissues, in the down regulation of Mn‐containing superoxide dismutase is discussed.