Evidence for active half-molecules of alpha 2-macroglobulin formed by dissociation in urea

Urea caused dissociation of alpha 2-macroglobulin (alpha 2M) into half-molecules (two disulfide-bonded subunits) as revealed by gel electrophoresis. The fraction of whole molecules remaining decreased with increasing urea concentration. Half-dissociation occurred at about 2.2 M. The ability of alpha 2M to inhibit trypsin also decreased with increasing urea concentration, but the activity-urea curve was shifted to the right as compared to the dissociation-urea curve. Thus, at 3 M urea, gel electrophoresis showed only 6.6% whole molecules, whereas the trypsin inhibitory activity was 95% of that in buffer with no urea, suggesting that half-molecules retain activity. In addition, complexes formed in urea with 125I-labeled trypsin were observed to migrate as half-molecules even though only 50% of such complexes were covalent. These results are surprising in light of the report by Gonias and Pizzo [Gonias, S., & Pizzo, S. (1983) Biochemistry 22, 536-546] that half-molecules formed by mild reduction are active; reduction is assumed to divide the molecule along an axis orthogonal to the break caused by urea. This suggests that active half-molecules can be formed by splitting either the covalent or noncovalent bonds that hold the subunits together. A model is proposed that can account for this possibility. It has the same dimensions and symmetry as a previous model of Feldman et al. [Feldman, S.R., Gonias, S.L., & Pizzo, S.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704] and accounts in a similar way for previous functional studies of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some alcohols on the conformation of mitochondrial H+-ATPase complex and F1-ATPase from pig heart

The conformations of the H+-ATPase complex and F1-ATPase in low concentrations of methanol, ethanol, n-propanol, iso-propanol and t-butanol were studied by circular dichroism. For F1-ATPase, all but methanol first increased and then decreased the circular dichroism magnitude of helical bands as the alcohol concentration was increased. With ethanol, n-propanol, iso-propanol and t-butanol, the alpha-helix content reached a maximum at about 5% alcohol and began to decrease at 10%. The content of beta-sheet showed the opposite effect, reaching a minimum at 5% and increasing slightly at higher concentrations. None of the alcohols studied had a significant effect on the conformation of the H+-ATPase complex. This difference implies that the alcohols had a greater effect on free F1-ATPase than on the membrane-bound F1-ATPase. The hydrophobic protein F0 and the membrane lipids in the H+-ATPase complex may stabilize and protect F1 from the effects of the alcohols.     

The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova

Fertilization and early development in the horse were studied by recovering oviductal ova at various times after postovulatory mating. Ova collected between 7 and 22 h post coitum (pc) were examined for evidence of fertilizing sperm, cellular changes accompanying fertilization, and pronuclear development. Five ova collected between 7 and 9 h pc contained a marginal metaphase plate, but had no indication of sperm components; three of these, however, showed reduced numbers of cortical granules. Two activated ova (10 and 14 h pc) were in telophase of the second meiotic division, following incorporation of the fertilizing sperm. The fertilizing sperm was situated in a slight elevation; the nucleus was expanding but lacked a nuclear envelope. The pronuclear stage in the horse began as early as 12 h pc, and lasted at least until 21 h pc. Sperm tail remnants were seen in 5 of 7 pronuclear-stage ova, although the crowding of the cytoplasm with clusters of lipid and vacuoles made discerning sperm tail remnants difficult. The spindles of the metaphase stage of the second meiotic division were oriented radially, that is, at right angles to the cell surface, in all but one ovum, so this orientation is not a response to fertilization.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton

The yeast tropomyosin gene, designated TPM1, is present in a single copy per haploid genome and encodes a protein with a predicted molecular weight of 23.5 kd. The protein sequence is homologous to higher cell tropomyosins, including the characteristic hydrophobic-hydrophilic pseudoheptapeptide repeats. Indirect immunofluorescence microscopy reveals that tropomyosin is localized with actin cables in wild-type cells. Disruption of TPM1 is not lethal, but results in a reduced growth rate and disappearance of actin cables. Strains carrying the conditional actin mutation act1-2 also lack actin cables; overexpression of tropomyosin in these strains partially restores actin cables. These results strongly suggest that tropomyosin interacts with F actin in vivo and may play an important role in assembling or stabilizing actin cables in yeast.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Diffusion of the Interspecies Electron Carriers H(2) and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of K(m) for H(2) or Formate Uptake

We calculated the potential H(2) and formate diffusion between microbes and found that at H(2) concentrations commonly found in nature, H(2) could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H(2) concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H(2) concentration at the cell surface of H(2)-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 mum from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K(m) for H(2) or formate.