Reconstitution of heme-synthesizing activity from ferric ion and porphyrins, and the effect of lead on the activity

We examined the activity of heme synthesis when ferrochelatase purified from rat liver mitochondria was incubated with ferric chloride and mesoporphyrin IX as substrates in the absence of reducing reagents. In the presence of the NADH dehydrogenase-rich fraction and NAD(P)H, mesoheme was synthesized; the addition of FMN or FAD markedly enhanced the activity. These results indicate that the NAD(P) H-oxidizing system reduces ferric ion to ferrous ion. This ferrous ion is then utilized for heme synthesis by ferrochelatase. The effect of lead on NAD(P)H-dependent heme synthesis was also examined. Lead reduced NAD(P)H-dependent heme synthesis by 50% at 10(-5) M, but had no effect when ferrous ion was used as substrate. Zn-Porphyrin synthesis was not changed in the presence of Pb2+ at 10(-5) M. Thus, heme synthesis from ferric ion was more susceptible to Pb2+ than heme synthesis from ferrous ion.     

Redox activity and peroxidase activity associated with the plasma membrane of guard-cell protoplasts

Redox systems have been reported in the plasma membrane of numerous cell types and in cells from various species of higher plant. A search for a redox system in the plasma membrane of guard cells was therefore made in efforts to explain how blue light stimulates stomatal opening, a process which is coupled to guard cell H⁺ efflux and K⁺ uptake. The rates of O₂ uptake by intact guard-cell protoplasts (GCP) of Commelina communis L., in the dark, were monitored in the presence of NAD(P)H since the stimulation of O₂ consumption by reduced pyridine nucleotides is used as an indicator of the presence of a redox system in the plasma membrane. Oxygen consumption by intact GCP increased two- to threefold in the presence of NAD(P)H. The NAD(P)H-stimulation of O₂ uptake was dependent on Mn²⁺ and was stimulated 10- to 15-fold by salicylhydroxamic acid (SHAM). Catalase, cyanide and ascorbate, a superoxide scavenger, all individually inhibited the SHAM-stimulated O₂ uptake. These are all characteristics of peroxidase activity although some of these features have been used to imply the presence of a redox system located in the plasma membrane. High levels of peroxidase activity (using guaiacol as a substrate) were also detected in the GCP and in the supernatant. The activity in the supernatant increased with time indicating that peroxidase was being excreted by the protoplasts. The properties of O₂ uptake by the incubation medium after separation from the protoplasts were similar to those of the protoplast suspension. It is concluded that our observations can be more readily explained by peroxidase activity associated with the plasma membrane and secreted by the GCP than by the presence of a redox system in the plasma membrane of the protoplasts.Abbreviations EDTA ethylenediaminetetraacetic acid - GCP guard cell protoplast - Mes 2-(N-morpholino)ethanesulphonic acid - SHAM salicylhydroxamic acid     

Cholesterol and the activity of bacterial toxins

Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin-cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureus alpha-hemolysin, aerolysin, and diphtheria toxin.     

Structure–activity relationship of celecoxib and rofecoxib for the membrane permeabilizing activity

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in terms of their structure–activity relationship, we separated the structures of celecoxib and rofecoxib into three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the membrane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl subgroup of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent or weak membrane permeabilizing activity, respectively. These findings provide important information for design and synthesis of new coxibs with lower membrane permeabilizing activity.     

Transport enhancement and the activity of insulin

The transport properties of insulin have been analysed with respect to the Brewer membrane model. Polar molecules are transported into the cell through their association with cations. The abilities of cations and their associated polar molecules to enter the cell membrane depend upon the activity of the cation and the quantum state of multivalent membrane bonds, principally the P = O located in the head of phospholipids. The active cation K forms an association with glucose and can enter the membrane when the bonds are in the ground state. Less active cations, such as Na, Ca and Mg, form larger and more complex associations but can only enter the membrane when the bonds are in an excited state. Insulin's effect on the transport of polar molecules is shown to correspond with its effect on the transport of cations. A possible model for insulin action on the cell membrane is explained.