Tetrathionate stimulated growth of Campylobacter jejuni identifies a new type of bi-functional tetrathionate reductase (TsdA) that is widely distributed in bacteria.

Tetrathionate (S4 O6 (2-) ) is used by some bacteria as an electron acceptor and can be produced in the vertebrate intestinal mucosa from the oxidation of thiosulphate (S2 O3 (2-) ) by reactive oxygen species during inflammation. Surprisingly, growth of the microaerophilic mucosal pathogen Campylobacter jejuni under oxygen-limited conditions was stimulated by tetrathionate, although it does not possess any known type of tetrathionate reductase. Here, we identify a dihaem cytochrome c (C8j_0815; TsdA) as the enzyme responsible. Kinetic studies with purified recombinant C. jejuni TsdA showed it to be a bifunctional tetrathionate reductase/thiosulphate dehydrogenase with a high affinity for tetrathionate. A tsdA null mutant still slowly reduced, but could not grow on, tetrathionate under oxygen limitation, lacked thiosulphate-dependent respiration and failed to convert thiosulphate to tetrathionate microaerobically. A TsdA paralogue (C8j_0040), lacking the unusual His-Cys haem ligation of TsdA, had low thiosulphate dehydrogenase and tetrathionate reductase activities. Our data highlight a hitherto unrecognized capacity of C. jejuni to use tetrathionate and thiosulphate in its energy metabolism, which may promote growth in the host. Moreover, as TsdA represents a new class of tetrathionate reductase that is widely distributed among bacteria, we predict that energy conserving tetrathionate respiration is far more common than currently appreciated.

Vanadate-induced Ca(2+) and Co(2+) uptake in human red blood cells.

The vanadate-induced increase in passive uptake of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. Vanadate is a potent inhibitor of the Ca-pump in red cells, although in fed cells a residual pump activity remains that is highly significant compared to the passive influx, and even in cells that are both ATP-depleted and vanadate-treated the pump arrest is not complete. In the presence of vanadate the Ca(2+) uptake is increased due to inhibition of Ca-pump extrusion, but is further increased due to a vanadate-induced increment in passive influx. In order to measure the vanadate-induced increment in Ca(2+) influx, the total uptake in vanadate-treated cells is corrected for the basal influx, as recorded in ATP-depleted cells in the presence of tetrathionate (5mM) that has been shown to eliminate the residual Ca-pump activity in ATP-depleted cells. The (57)Co uptake is also increased by vanadate. (57)Co is not transported by the Ca-pump, and hence the uptake in vanadate-treated cells can be directly compared to the basal uptake, both in fed and in ATP-depleted cells. The vanadate effect shows rapid onset and appears to be irreversible. The vanadate-induced increment in uptake of both (45)Ca and (57)Co is reduced by about 50% in ATP-depleted cells compared to fed cells, suggesting a metabolism- or SH-group-dependent component. The influx of both (45)Ca (in ATP-depleted cells) and (57)Co (in fed cells) increases with the vanadate concentration, with a similar K(½) (0.4 and 0.3mM, respectively), and is nearly maximal at 5mM vanadate. The vanadate-induced increment in influx of both (45)Ca and (57)Co increases with the extracellular concentration as a saturable function, with K(½) estimated at, respectively, 700 and 80µM. In the case of (57)Co K(½) is similar in fed and in ATP-depleted cells. The vanadate-induced uptake of (45)Ca and of (57)Co shows interference. The uptake of (45)Ca is inhibited by Co(2+), and the uptake of (57)Co is inhibited by Ca(2+), although with an unexplained time course. The vanadate-induced uptake of (45)Ca and (57)Co are both inhibited, and to a similar degree, by the 1,4-dihydropyridine Ca(2+)-channel blocker nifedipine, although only at concentrations much higher than IC(50) for classical Ca-channels. The vanadate-induced increment in (57)Co uptake is electroneutral, in contrast to the basal uptake that is at least partially electrogenic. In experiments with resealed ghosts a vanadate-induced (57)Co uptake could not be detected. The vanadate-induced increment in (57)Co uptake amounts to nearly half the increment in (45)Ca uptake, both in fed and in ATP-depleted cells. It is speculated that the vanadate-induced Ca(2+) and Co(2+) uptake could be mediated by a putative common transporter, which appears to be separate and distinct from the putative common transporter for basal Ca(2+) and Co(2+) uptake.

Passive transport pathways for Ca(2+) and Co(2+) in human red blood cells. (57)Co(2+) as a tracer for Ca(2+) influx.

The passive transport of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1µM, the progress curve for (45)Ca uptake at 1mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca(T)](c) to reach at [Ca(T)](c) about 5µmol(lcells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca(T)](c). Moreover, vanadate markedly increases the passive Ca(2+) influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40-50µmol(lcells)(-1)h(-1). The apparent Ca(2+) affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the (45)Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1mM Ca(2+) estimated at 50-60µmol(lcells)(-1)h(-1). The Ca influx increases with the extracellular Ca(2+) concentration with a saturating component, with K(½(Ca)) about 0.3mM, plus a non-saturating component. From (45)Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The (45)Ca efflux is markedly accelerated by external Ca(2+), both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The (57)Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co(2+) is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The (57)Co uptake shows several similarities with the (45)Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J(max) about 16µmol(lcells)(-1)h(-1) and K(½(Co)) about 0.1mM, plus a non-saturating component. The (57)Co and (45)Ca uptake shows mutual inhibition, and at least the stochastic Ca(2+) influx is inhibited by Co(2+). The (57)Co and (45)Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100µM. The (57)Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The (57)Co influx amounts to about half the (45)Ca influx in ATP-depleted cells. It is speculated that the basal Ca(2+) and Co(2+) uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that (57)Co could be useful as a tracer for at least the channel-like Ca(2+) entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca(2+) buffering. The molecular identity of the putative common transporter(s) remains to be defined.

Relation between the organization of spectrin and of membrane lipids in lymphocytes.

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.

Thiol-dependent membrane transport of selenium through an integral protein of the red blood cell membrane.

The molecular details of the selenium metabolism and transport in living systems are still not completely understood, despite their physiological importance. Specifically, little is known about the membrane transport of selenium from most of the selenium containing compounds. In the present study, we investigated the mechanism for the membrane transport of selenium from red blood cells (RBCs) to the blood plasma. When the selenium distribution in the RBC ghost membrane after treatment with selenious acid was analyzed, nearly 70% of the selenium in the membrane was found to bind to the anion exchanger 1 (AE1) protein, which suggested that the integral protein AE1 is responsible for the membrane transport of selenium. The thiol dependency of the selenium export from the RBC to the blood plasma was examined using membrane permeable thiol reagents, i.e., N-ethylmaleimide (NEM) and tetrathionate (TTN). Treatment of the RBC with NEM, a thiol-alkylating reagent, resulted in modification of the thiol groups in the amino-terminal cytoplasmic domain (N-CPD) of the AE1, but not those in the membrane domain. Such an NEM treatment provided a marked inhibition of the selenium export from the RBC to the blood plasma. In addition, the treatment with TTN, a thiol-oxidizing reagent that forms intermolecular disulfide bonds, appeared to oxidize thiol groups in both the N-CPD and the membrane domain of AE1, which resulted in complete inhibition of the selenium export even during the initial period in which the export had a maximum velocity when using the thiol reagent-free treatment. Such complete inhibition of the selenium export from the TTN-treated RBC appeared to be due to the oligomerized AE1 proteins resulting from the intermolecularly formed disulfide bonds. These inhibitory effects using NEM and TTN suggested that thiol groups in the integral protein AE1 play essential roles in the membrane transport of the selenium from the RBCs to the blood plasma.

Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle.

Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite).

N-terminal insertion of alamethicin in channel formation studied using its covalent dimer N-terminally linked by disulfide bond.

Alamethicin is supposed to form helix-bundle-type channels by inserting the N terminus into bilayer lipid membranes under sufficient voltages. The N-terminal insertion has been studied with an alamethicin dimer (di-alm) N-terminally linked by a disulfide bond and by the asymmetric addition of dithiothreitol (DTT) and tetrathionate (TT) to the membrane. When di-alm was added to the cis-side membrane, it forms long-lasting channels with the lifetime tau of about 100 ms at cis-positive voltages. The lifetime was reduced to a few milliseconds by addition of DTT to the cis-side membrane, indicating that most of the channels were formed by the monomers (alm-SH) that resulted from the cleavage of the disulfide bond in di-alm. The succeeding addition of TT to the trans-side produced channels of tau=10-20 ms besides the channels of alm-SH. The results suggested that TT reacted with the N-terminal thiol group of alm-SH located at the trans-side of the membrane to alter the lifetime. The N-terminal insertion of alamethicin helices by voltage activation, therefore, was confirmed.

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane.

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association.

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.

Showdomycin, a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase.

Showdomycin [2-(beta-D-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na+ + K+)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01-mol- minus 1-min- minus 1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K+, 100 MUM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na+ + K+)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase and inhibiton is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.

Stimulatory and inhibitory effects of sulfhydryl reagent on p-aminohippuric acid transport by isolated renal tubules.

Isolated tubules from rabbit kidney cortex were treated with several different sulfhydryl reagents in an attempt to determine whether sulfhydryl groups are involved in organic acid transport. Disulfide reagents such as sodium tetrathionate and 6,6'-dithionicotinic acid were found to exert a biphasic effect on p-aminohippuric acid transport, i.e. transient stimulation followed by inhibition. In contrast, treatment of tubules with the mercaptide-forming reagent, p-chloromercuribenzoate, caused only inhibition of organic acid transport. Treatment of tubules with reductants such as dithiothreitol or mercaptoethanol blocked the stimulatory effect of tetrathionate without affecting the inhibitory effect of this oxidant. The inhibition caused by p-chloromercuribenzoate, however, was largely reversible when tubules were treated with reductants. The results suggest that the renal organic acid transport system contains sulfhydryl groups and that its activity is increased when some of these groups are oxidized.

Experimental alteration of phospholipid-protein interactions within the human erythrocyte membrane. Dependence on glycolytic metabolism.

Phosphatidylethanolamine in freshly drawn human erythrocytes is trinitrophenylated by 2,4,6-trinitrobenzene sulfonic acid only slowly and to a maximum of 32%. After different preincubation procedures at 37 degrees C in saline media in the absence of glucose (24 h without additive, 1-5 h with 8 mM hexanol or 1-4 h with the SH reagent, 5 mM tetrathionate) the rate of subsequent trinitrophenylation of phosphatidylethanolamine, in the absence of the additives, is greatly enhanced and the amount of phospholipid reacting increased. Glucose or inosine prevent these effects, inhibitors of glycosis abolish this protection. The results indicate that in fresh as well as in glycolysing incubated erythrocytes phosphatidylethanolamine in the outer layer of the membrane lipid is shielded by a protein. Conformational changes of this protein induced by metabolic starvation and perturbing agents expose the phospholipid head group to 2, 4, 6-trinitrobenzene sulfonic acid. In addition, a "flip-flop" of phosphatidylethanolamine from the inner to the outer layer may also contribute to the effects observed.

Intra- and intermolecular cross-linking of membrane proteins in intact erythrocytes and ghosts by SH-oxidizing agents.

In intact human erythrocytes, SH-oxidizing agents exclusively cross-link spectrin via disulfide bonds. In ghosts, additional dimerization of the major intrinsic protein, band 3, is observed. After blockade of intracellular GSH the agents dimerize band 3 in the intact cell too, indicating that GSH may prevent band 3 dimerization under physiological conditions. The oxidizing agents reversibly oxidize 80% of the membrane SH-groups, suggesting that these groups are arranged close enough to each other to form disulfide bonds. This arrangement may protect other cell cell structures against free radicals or oxidative stress.

Complete exchange of phosphatidylcholine from intact erythrocytes after protein crosslinking.

Treatment of human erythrocytes with tetrathionate or diamide resulted in extensive crosslinking of membranous and cytoskeletal proteins. Such treatment was followed by an incubation with phosphatidylcholine specific exchange protein to investigate the rate and extent of exchange of phosphatidylcholine between the erythrocytes and 14C-labeled phosphatidylcholine containing microsomal membranes or vesicles. Exchange profiles showed that the exchange of phosphatidylcholine is facilitated in treated cells when compared to control erythrocytes and, more importantly, that all of the phosphatidylcholine is exchangeable after protein crosslinking whereas in control cells only the phosphatidylcholine pool located in the outer layer of the membrane is exchangeable. These observations demonstrate that crosslinking of cytoskeletal and membraneous proteins enhances the rate of transbilayer movement of phosphatidylcholine considerably.

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects on the sulfur-free and sulfur-containing catalytic intermediates.

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave Km (Ks) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reversed by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.

A new form of inactive renin in rat brain. A latent renin.

A new inactive (latent) form of renin was found in rat brain extract. It is activated by sulfhydryl compounds such as dithiothreitol but not by proteases such as trypsin. The activated form of latent renin in crude brain extract was again inactivated by the disulfide compound sodium tetrathionate. Latent renin was separated, at least partially, from active renin by affinity chromatography on Affi-Gel Blue. In contrast to a marked (10-fold) increase of latent renin by dithiothreitol, the enzyme activity of active renin was increased by less than 50% by this sulfhydryl compound. Thus, the major part of the activating effect of dithiothreitol does not seem to be due to its effect on renin substrate. Latent renin showed affinity for pepstatin-Sepharose gel. These properties indicate that latent renin is different from inactive renin of the zymogen type, which is activated by protease or acid treatment but not by sulfhydryl compounds and does not show affinity to pepstatin. Latent renin has a molecular weight of 45,000 and is reduced to 34,000 upon activation by dithiothreitol. This observation suggests that latent renin may be a renin-inhibitor complex.

L1210 dihydrofolate reductase: activation and enhancement of methotrexate sensitivity.

Dihydrofolate reductase, purified to homogeneity from a subline of L1210 murine leukemia cells resistant to 10(-6) M Methotrexate, was resolved into two principal forms (1 and 2) by isoelectric focusing. These forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. Form 1 was a single component, comprising ca. 10% of the total protein, but multiple components of 2 were observed by narrowing the pH range in the electrophoretic procedure. Urea-denatured enzyme exhibited two bands of approximately equal intensity upon isoelectric focusing. These results were interpreted to mean that the enzyme consists of a set of conformationally different forms, arising from two primary structures. Inhibition of the native enzyme by Methotrexate was polyphasic, and appreciable activity remained when the drug was present at an equimolar concentration. Various agents (KCl, H+, urea, and SH-modifiers), known to "activate" dihydrofolate reductases, produced a monophasic, stoichiometric inhibition. Activating agents appear to induce a more open (and labile) conformation of the enzyme. This leads to increased affinity for MTX accompanied, in some instances, by increased catalytic activity.

Tetrathionate-blocked creatine kinase as a substrate for human plasma 'creatine kinase conversion factor'.

'Creatine kinase conversion factor' has been partially purified from whole plasma. The preparation has the ability to convert slowly migrating, native CK-MM into a faster electrophoretic form through a form of intermediate mobility. These changes are accompanied by a loss in CK activity. The rates of both electrophoretic conversion and loss of enzyme activity are reduced or prevented by the presence of 2-mercaptoethanol, EDTA, or by formation of the creatine-MgADP-nitrate dead-end complex. When the thiol group essential for enzyme activity found at the CK active site is first blocked by treatment with potassium tetrathionate, full activity may be retained under conditions that fully convert CK to the faster electrophoretic forms. The conversion factor activity was not inhibited by a pre-incubation with the protease inhibitor, PMSF and the converted form of CK showed no evidence of proteolytic cleavage. The molecular basis of the chemical alteration caused by the conversion is discussed.

[Possibility of decreasing the anticomplement activity of human immunoglobulin preparations via chemical modification]. / Vozmozhnost' snizheniia antikomplementarnoi aktivnosti preparatov immunoglobulina cheloveka s pomoshch'iu khimicheskoi modifikatsii.

The physicochemical and immunological properties of the experimental batches of the preparations of placental immunoglobulin, obtained by some methods of chemical modification of the molecule of IgG, have been studied. The possibility of abolishing the anticomplement properties of the preparations treated with sulfitolytic agents manufactured in the USSR has been shown. The optimum conditions permitting the production of the preparation with faintly pronounced anticomplement properties and the full monomer structure of its molecule have been established.