Comparison of outcomes between clipping and endovascular coiling in anterior choroidal artery aneurysm: a systematic review.

SAH (subarachnoid hemorrhage) caused by aneurysm rupture has the greatest mortality rate, with nearly 50% of patients unable to survive beyond 1 month after the attack. Anterior choroidal artery (AChA) aneurysms are one of the most difficult to treat among the numerous types of aneurysms. Until now, some neurosurgeons employed shearing while others employed coiling. In this trial, researchers will compare surgical clipping and endovascular coiling treatments for anterior choroidal artery aneurysms in terms of mortality, rebleeding, retreatment, and post-procedure outcomes. Using the PubMed electronic database, the Cochrane library, the Medline Database, the Directory of Open Access Journals, and EBSCHOHOST, a systematic review compared surgical clipping and endovascular coiling in all cases of choroidal artery aneurysm. There were 17 studies that met the eligibility requirements, with a total of 1486 patients divided into groups that underwent clipping (1106) or endovascular coiling (380). The mortality rate for clipping is 1.8%, while the mortality rate for endovascular coiling is 2.34%. Rebleeding occurs in 0% of patients undergoing endovascular coiling and 0.73% of patients undergoing clipping. Retreatment of clipping was 0.27%, while endovascular coiling was 3.42%. Post-complication procedures occurred in 11.12% of patients undergoing endovascular clipping and 15.78% of patients undergoing endovascular coiling. The intervention technique of clipping has a reduced rate of mortality, reoperation, and post-operative complications. Endovascular coiling results in a reduced rate of rebleeding than clipping.

Potential role of flavonoids against SARS-CoV-2 induced diarrhea.

COVID-19, caused by the SARS-CoV-2 virus, can lead to massive inflammation in the gastrointestinal tract causing severe clinical symptoms. SARS-CoV-2 infects lungs after binding its spike proteins with alveolar angiotensin-converting enzyme 2 (ACE2), and it also triggers inflammation in the gastrointestinal tract. SARS-CoV-2 invades the gastrointestinal tract by interacting with Toll-like receptor-4 (TLR4) that induces the expression of ACE2. The influx of ACE2 facilitates cellular binding of more SARS-CoV-2 and causes massive gastrointestinal inflammation leading to diarrhea. Diarrhea prior to COVID-19 infection or COVID-19-induced diarrhea reportedly ends up in a poor prognosis for the patient. Flavonoids are part of traditional remedies for gastrointestinal disorders. Preclinical studies show that flavonoids can prevent infectious diarrhea. Recent studies show flavonoids can inhibit the multiplication of SARS-CoV-2. In combination with vitamin D, flavonoids possibly activate nuclear factor erythroid-derived-2-related factor 2 that downregulates ACE2 expression in cells. We suggest that flavonoids have the potential to prevent SARS-CoV-2 induced diarrhea.

In silico screening of Allium cepa phytochemicals for their binding abilities to SARS and SARS-CoV-2 3C-like protease and COVID-19 human receptor ACE-2.

Corona virus SARS-CoV-2-induced viral disease (COVID-19) is a zoonotic disease that was initially transmitted from animals to humans. The virus surfaced towards the end of December 2019 in Wuhan, China where earlier SARS (Severe Acute Respiratory Syndrome) had also surfaced in 2003. Unlike SARS, SARS-CoV-2 (a close relative of the SARS virus) created a pandemic, and as of February 24 2021, caused 112,778,672 infections and 2,499,252 deaths world-wide. Despite the best efforts of scientists, no drugs against COVID-19 are yet in sight; five vaccines have received emergency approval in various countries, but it would be a difficult task to vaccinate twice the world population of 8 billion. The objective of the present study was to evaluate through in silico screening a number of phytochemicals in Allium cepa (onion) regarding their ability to bind to the main protease of COVID-19 known as the 3C-like protease or 3CLpro, (PDB ID: 6LU7), 3CLpro of SARS (PDB ID: 3M3V), and human angiotensin converting enzyme-2 (ACE-2), [PDB ID: 1R42], which functions as a receptor for entry of the virus into humans. Molecular docking (blind docking, that is docking not only against any target pocket) were done with the help of AutoDockVina. It was observed that of the twenty-two phytochemicals screened, twelve showed good binding affinities to the main protease of SARS-CoV-2. Surprisingly, the compounds also demonstrated good binding affinities to ACE-2. It is therefore very likely that the binding affinities shown by these compounds against both 3CLpro and ACE-2 merit further study for their potential use as therapeutic agents.

Can Costus afer be used for co-treatment of COVID-19, its symptoms and comorbidities? A novel approach for combating the pandemic and implications for sub-Saharan Africa.

Despite the huge loss of lives and massive disruption of the world economy by the COVID -19 pandemic caused by SARS -CoV-2, scientists are yet to come out with an effective therapeutic against this viral disease . Several vaccines have obtained 'emergency approval ', but difficulties are being faced in the even distribution of vaccines amongst high- and low- income countries . On top of it, comorbidities associated with COVID -19 like diabetes, hypertension and malaria can seriously impede the treatment of the main disease, thus increasing the fatality rate . This is more so in the context of sub -Saharan African and south Asian countries . Our objective was to demonstrate that a single plant containing different phytoconstituents may be used for treatment of COVID -19 and comorbidities . Towards initial selection of a plant, existing scientific literature was scanned for reported relevant traditional uses, phytochemicals and pharmacological activities of a number of plants and their phytoconstituents pertaining to treatment of COVID-19 symptoms and comorbidities. Molecular docking studies were then performed with phytochemicals of the selected plant and SARS-CoV-2 components - Mpro, and spike protein receptor binding domain and hACE2 interface using AutoDock V ina. We showed that crude extracts of an indigenous African plant, Costus afer having traditional antidiabetic and antimalarial uses, has phytochemicals with high binding affinities for Mpro, and /or spike protein receptor binding domain and hACE2 interface; the various phytochemicals with predicted high binding energies include aferoside C, dibutyl phthalate, nerolidol, suginal, and ± -terpinene, making them potential therapeutics for COVID -19. The results suggest that crude extracts and phytochemicals of C. afer can function as a treatment modality for COVID -19 and comorbidities like especially diabetes and malaria .

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia.

This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete ß-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 104, 106, and 108 CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD50-336 h of S. iniae against the red hybrid tilapia was 102 CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia. Received January 20, 2017; accepted July 16, 2017.

Synergistic regulation of Schwann cell proliferation by heregulin and forskolin.

A peptide corresponding to the epidermal growth factor homology domain of beta-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95(RSK2), was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Differential coupling of alpha1-adrenoreceptor subtypes to phospholipase C and mitogen activated protein kinase in neonatal rat cardiac myocytes.

Activation of cardiac alpha1-adrenoreceptors has a number of physiological effects. Ascribing these effects to a specific alpha1-adrenoreceptor subtype first requires the elucidation of the subtypes that are present in the tissue of interest. In the present study, mRNA transcripts for the alpha1A, alpha1B and alpha1D-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using reverse transcriptase-polymerase chain reaction analysis. However, binding sites for only the alpha1A and alpha1B-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using competition binding analysis with a variety of alpha1 selective receptor antagonists. Phenylephrine-stimulated phosphatidylinositol hydrolysis was inhibited by alpha1 selective receptor antagonists with affinities consistent with the alpha1A-adrenoreceptor subtype, whereas phenylephrine-induced activation of the mitogen activated protein kinase cascade was inhibited by these same antagonists with affinities more closely resembling the alpha1B-adrenoreceptor subtype. In the case of both signaling pathways, the alpha1D selective receptor antagonist, BMY 7378, exhibited affinities suggestive of the relative absence of a alpha1D-adrenoreceptor subtype. Thus, despite the presence of mRNA transcripts for all three alpha1-adrenoreceptor subtypes, only the alpha1A and alpha1B-adrenoreceptor subtypes were expressed and functionally coupled at detectable levels in neonatal rat cardiac myocytes. Of particular interest, phenylephrine-induced activation of the mitogen activated protein kinase cascade appears to be mediated by a subtype resembling most closely the pharmacological profile of the alpha1B-adrenoreceptor subtype.

Enzymatic hydrolysis of long-chain N-heterocyclic fatty esters.

Incubation of a 1-pyrroline ester [viz. methyl 8-(5-hexyl-1-pyrroline-2-yl)octanoate, 1] with bakers' yeast (Saccharomyces cerevisiae) gave the corresponding free fatty acid (1a, 52%). The C = N bond of the 1-pyrroline was not reduced by the yeast. Complete hydrolysis of compound 1 was successful using lipase of Candida cylindracea (CCL) or Lipolase (Rhizomucor miehei) under stirred or ultrasound condition. Fatty esters containing a pyrrolidine [viz. methyl 8-(cis/trans-5-hexyl-pyrrolidine-2-)octanoate, 2] or N-methyl pyrrolidine [viz. methyl 8-(cis-5-hexyl-N-methyl-pyrrolidine-2-)octanoate, 3] system in the alkyl chain were not hydrolyzed by either CCL or Lipolase, unless conducted in an ultrasonic bath. The hydrolytic activities of the enzymes appeared to be strongly affected by the stereochemistry of the N-heterocyclic ring system. Chemical hydrolysis of compounds 1-3 gave the corresponding fatty acid N-HCl salts.

Specificity of G protein alpha-gamma subunit interactions. N-terminal 15 amino acids of gamma subunit specifies interaction with alpha subunit.

The existence of multiple alpha, beta, and gamma subunits raises questions regarding the assembly of particular G proteins. Based on the results of a previous study (Rahmatullah, M., and Robishaw, J. D. (1994) J. Biol. Chem. 269, 3574-3580), we hypothesized that the assembly of G proteins may be determined by the interactions of the more structurally diverse alpha and gamma subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the gamma 1 and gamma 2 subunits to interact with the alpha o subunit. Chimeras of the gamma 1 and gamma 2 subunits were used to delineate which region is responsible. Support for the importance of the N-terminal region of the gamma subunit comes from our observations that 1) the gamma 2 subunit and the gamma 211 chimera bound strongly to the alpha o-agarose matrix, but the gamma 1 subunit and the gamma 112 chimera bound weakly, if at all; 2) an N-terminal peptide made to the gamma 2 subunit blocked the binding of the gamma 211 chimera to the alpha o-agarose matrix; 3) both the gamma 211 chimera and the N-terminal peptide were able to partially protect the alpha o subunit against tryptic cleavage; and 4) the gamma 211 chimera, but not the gamma 112 chimera, supported ADP-ribosylation of the alpha o subunit.

Chemical and enzymatic preparation of acylglycerols containing C18 furanoid fatty acids.

C18 furanoid triacylglycerol [glycerol tri-(9,12-epoxy-9,11-octadecadienoate)] was prepared by chemical transformation of triricinolein isolated from castor oil. The procedure involved oxidation, epoxidation and cyclization of the epoxy-keto intermediate with sodium azide and ammonium chloride in aqueous ethanol. The furanoid triacylglycerol was also obtained by esterification of C18 furanoid fatty acid with glycerol using Novozyme 435 (Novo Nordisk A.S., Bagsvaerd, Denmark) as biocatalyst. When Lipozyme (Novo Nordisk A.S.) was used, a mixture of the furanoid 1(3)-rac-monoacylglycerol and 1,3-diacylglycerol was obtained. In order to obtain the C18 furanoid 1,2(2,3)-diacylglycerol, selective hydrolysis of the furanoid triacylglycerol was achieved using porcine pancreatic lipase in tris(hydroxymethyl) methylamine buffer. Interesterification of triolein with methyl C18 furanoid ester in the presence of Lipozyme showed maximum incorporation of 34% of furanoid fatty acid. Extension of the interesterification to vegetable oils (olive, peanut, sunflower, corn and palm oil) allowed a maximum of 24% furanoid acid incorporation to be achieved.

Ultrasound in fatty acid chemistry: synthesis of a 1-pyrroline fatty acid ester isomer from methyl ricinoleate.

A novel 1-pyrroline fatty acid ester isomer (viz. 8-5-hexyl-1-pyrrolin-2-yl) octanoate) has been synthesized from methyl ricinoleate by two routes with an overall yield of 42 and 30%, respectively. Most of the reactions are carried out under concomitant ultrasonic irradiation (20 KHz, ca. 53 watts/cm2). Under such a reaction condition, the reaction time is considerably shortened, and product yields are high. Dehydrobromination under concomitant ultrasonic irradiation of methyl 9, 10-dibromo-12-hydroxyoctadecanoate with KOH in EtOH furnishes methyl 12-hydroxy-9-octadecynoate (66%) within 15 min. Hydration of the latter under ultrasound with mercury(II)acetate in aqueous tetrahydrofuran yields exclusively methyl 12-hydroxy-9-oxo-octadecanoate (95%) in 30 min. The hydroxy group in the latter compound is transformed to the azido function via the mesylate, and treatment of the azido-oxo intermediate (methyl 12-azido-9-oxooctadecanoate) with Ph3P under ultrasonic irradiation furnishes the requisite 1-pyrroline fatty acid ester (77%). The same azido-oxo intermediate has also been obtained by the oxidation of methyl 12-azido-9-cis-octadecenoate using benzoquinone and a catalytic amount of Pd(II)chloride in aqueous tetrahydrofuran under concomitant ultrasonic irradiation (90 min) to give the product in 45% yield. The latter reaction does not take place even under prolonged silent stirring of the reaction mixture.

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis.

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.

Partial activation of the pyruvate dehydrogenase kinase by the lipoyl domain region of E2 and interchange of the kinase between lipoyl domain regions.

The binding of the pyruvate dehydrogenase (E1) component and the E1-specific kinase to the core-forming dihydrolipoyl acetyltransferase (E2) component facilitates a severalfold enhancement in the rate at which the kinase phosphorylate E1 (i.e. versus free kinase phosphorylating free E1). The kinase and E1 associate with small exterior linker region-connected domains in the E2 structure. The kinase binds to one of two lipoyl domains, and the E1 component binds to a domain in E2"s structure between the lipoyl domain region and the inner domain. Sixty of the latter domains assemble to form a dodecahedron-shaped inner core. Binding of the kinase to a detached lipoyl domain region enhanced kinase activity. This bi-lipoyl domain fragment induced a 2-fold enhancement in the slow rate of phosphorylation of peptide substrate and intact E260 gave only a 50% higher rate. In contrast, the lipoyl domain fragment gave only a 40% enhancement in the faster rate of phosphorylation of E1; whereas the rate of phosphorylation of E1 was markedly increased (4-10-fold depending on conditions) by kinase interaction with the intact E2 core. Binding of E1 to an E2 structure lacking only the bi-lipoyl domain region did not enhance kinase activity. Thus, binding of the kinase to the lipoyl domain region elicits a structural change which enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected by intact E2 core. Among the latter is a need for a mechanism allowing one kinase molecule to phosphorylate many E1 tetramers, whereas both E1 and the kinase stay bound to the oligomeric E2 core (i.e. phosphorylation appears to be much faster than the dissociation of either the kinase or E1 tetramers from E260 core). Exposure of kinase bound to the lipoyl domain fragment to intact E2 core for 10 s allowed a transition to a maximal (7-fold) activation of the kinase. In the opposite direction, an increasing level of the free bi-lipoyl domain fragment rapidly reduced, in a concentration-dependent manner the activity of kinase bound initially to intact E2. The data strongly support kinase transfer between free lipoyl domains and the intact E2 core and fit about a 12-fold tighter binding of the kinase to intact E2 cores over binding to free lipoyl domains. Such an interchange of the kinase between these E2 structures was confirmed by sucrose gradient studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a cAMP-independent Ca2(+)-inhibited protamine kinase from Candida lipolytica.

A cAMP-independent protamine kinase has been purified from extracts of the yeast Candida lipolytica by ion-exchange and affinity chromatography. Two subunits with apparent Mr's of 52,000 and 36,000 were resolved by SDS-PAGE. The purified kinase exhibited about 20% activity with casein and histone Type VII-S as substrates relative to protamine. The enzyme was inactive against other protein substrates tested, and was essentially insensitive to AMP, cAMP, cGMP up to 0.2 mM, the polyamines spermine and spermidine up to 1 mM, N-ethylmaleimide (5 mM), 2-mercaptoethanol (20 mM), or dithiothreitol (2 mM), and several cations like Zn2+, N1+, or Co2+ at 0.1 mM each. Ca2+ at 3 mM inhibited protamine kinase activity by 50%, which was reversed by EGTA.

Dinitrochlorobenzene therapy in alopecia areata.

With topical therapy using dinitrochlorobenzene (DNCB), 36 (72%) out of 50 patients having alopecia areata showed regrowth of hair in 3 to 6 months time. Five (10%) patients experienced marked vesiculation and severe contact dermatitis requiring cessation of therapy. In 7 (14%) patients there was no response at all and 4 (8%) presented with other side-effects.

Changes in the core of the mammalian-pyruvate dehydrogenase complex upon selective removal of the lipoyl domain from the transacetylase component but not from the protein X component.

The dihydrolipoyl transacetylase (E2) component contains a COOH-terminal inner domain (E2I) and an extended NH2-terminal structure, which is composed of two lipoyl domains (the fragment containing both is designated as E2L) and a subunit-binding domain (E2B). The four domains are connected by hinge regions. A subcomplex, composed of an oligomer of E2 subunits, protein X (which also has an NH2-terminal lipoyl domain), and the [pyruvate dehydrogenase]-kinase catalytic and basic subunits (Kc and Kb, respectively) (i.e. E2.X.KcKb subcomplex), was treated with Clostridium histolyticum collagenase. E2 subunits were selectively cleaved at the NH2-terminal end of the E2B domain, releasing the E2L fragment. Complete release of E2 subunits also released the kinase subunits, indicating that the kinase is bound to the E2L portion of E2. The residual inner core subcomplex (designated E2IB.X) has a strong tendency to aggregate, but this can be reversed with heparin (1 mg/ml). The E2IB.X subcomplex binds the pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components. The E1 component, which binds to the E2B domain, blocked collagenase cleavage of E2. We evaluated the capacity of the collagenase-treated E2.X.KcKb subcomplex, from which different portions of the E2L domains were removed, to support (in combination with excess levels of the E1 and E3 components) the overall reaction of the complex. Loss of activity occurred only after more than half of the E2L domains were removed. This delay is in sharp contrast to the effect of selective removal of the lipoyl domain of protein X, which leads to an immediate decrease in activity (Gopalakrishnan, S., Rahmatullah, M., Radke, G.-A., Powers-Greenwood, S. L., and Roche, T. E. (1989) Biochem. Biophys. Res. Commun. 160, 715-721). These results suggest that multiple lipoyl domains of the E2 component service the rate-limiting E1 component. After all the E2L domains were removed and the E2IB.X subcomplex was separated from free E2L, 10% activity was retained in the overall reaction. Thus, the lipoyl domain of protein X supported the overall reaction of the complex.

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Separation of protein X from the dihydrolipoyl transacetylase component of the mammalian pyruvate dehydrogenase complex and function of protein X.

The dihydrolipoyl transacetylase (E2)-protein X-kinase subcomplex was resolved to produce an oligomeric transacetylase that was free of protein X and kinase subunits. We investigated the properties of this transacetylase E2 oligomer and of a form of the subcomplex from which only the lipoyl-bearing domain of protein X (XL) was removed. While retaining other catalytic and binding properties of the native subcomplex, the oligomeric transacetylase and the subcomplex lacking the XL domain had greatly reduced capacities both to support the overall reaction of the complex (upon reconstitution with other components) and to bind the dihydrolipoyl dehydrogenase component. Our results indicate that protein X, in part through its XL domain, contributes to the binding of the dihydrolipoyl dehydrogenase component and to the overall reaction of the complex.

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.