In the present study, the authors of this paper mainly used limited tryptic proteolysis to explore and probe the subunit structure of dihydrolipoyl transacetylase of bovine heart. The results of the paper have demonstrated that the subunit of bovine heart dihydrolipoyl transacetylase consists of two different folding domains designated the subunit-binding domain(Mr≈26,000) and the lipoyl domain(Mr≈28,600), respectively. They also concluded that the compact subunit-binding domain which gives rise to transacetylase’s quaternary structure is attached to the extended lipoyl domain bearing a covalently bound lipoyl moiety. Based on what we have known, the mammalian pyruvate dehydrogenase complex (PDC) is composed of three functional enzyme components: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Because E2 plays a pivotal role among these three components of PDC in catalyzing the reactions leading to the conversion of pyruvate to acetyl-CoA, its subunits composition and crystalline structure are essential to understand the overall reactions of PDC which connects glycolysis and citric acid cycle in biological processes. Additionally, since “there is discrepancy between molecular weights of transacetylase subunits determined by sodium dodecyl sulfate gel electrophoresis and by sedimentation equilibrium” (10) in different organisms (e.g. E.coli and mammals), the authors of this paper conducted a serious of experiments to confirm the precise subunit structure of E2 of bovine heart.
Products Identification from Limited Tryptic Digestion of E2
In this section, samples of purified E2 isolated from bovine heart were digested by trypsin. The limited proteolysis was stopped by adding excess soybean trypsin inhibitor, and after digestion the incubation mixtures were analyzed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 showed that with the digestion time increased, the transacetylase subunit was eventually cleaved into two major tryptic fragments designated L and B with apparent Mr≈37,000 and 28,000. However, Figure1 did not illustrate any gel lanes of transacetylase subunit which was not subjected to limited tryptic digestion. The reason why I think there is a missing control here simply because that as mentioned in the paper, the authors found the purified samples of E2 may produce a different minor fragment cleaved by an endogenous protease. This will lead to an incorrect analysis of products produced by exogenous trypsin digestion. Thus, to more precisely distinguish the cleavage products of E2 by tryptic digestion, the experiment should add this control.
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In order to further identify these two major fragments produced from limited tryptic digestion, the lipoyl moieties of E2 were labeled with [2-14C] pyruvate to track its radioactivity. After incubation of the mixture in the presence of thiamin pyrophosphate (TPP), E1 and N-ethylmaleimide, the lipoyl moieties were reductively acetylated and the products containing the labeled moieties and subsequently digested by trypsin from E2 were analyzed by SDS-PAGE again. The results showed that a radioactive fragment (termed “lipoyl domain”) was released with apparent Mr≈37,000 and by using ultracentrifugation or gel filtration, it was separated from the trypsin-modified transacetylase. Meanwhile, the major component of the modified transacetylase precipitated as a pellet was a polypeptide designated tryptic fragment B with apparent Mr≈28,000.
Trypsin Proteolysis of Bovine Heart PDC
Same methods and techniques were used here to analyze the trypsin digested products of bovine heart PDC and control samples from E1 and E2. The gel patterns of Figure 2 demonstrated that as the presence of both E1 and E3 can protect E2 from proteolysis, the cleavage rate of the transacetylase subunits in PDC was slower than in isolated transacetylase. In my opinion, the authors draw the conclusion based on the observations from Figure 2 that in gel lanes of limited tryptic digestion of PDC, there was no cleavage of E2 throughout the proteolysis process. In contrast, the sample of isolated E2 was cleaved by trypsin into two major fragments as noted above. However, I think this experiment’s results would be more convincing if they add another control sample of E3 similarly digested with trypsin. This additional control may not only support the authors’ claim that E3 was stable to trypsin digestion under the indicated conditions but also provide evidence to explore whether E3’s cleavage would be affected by other two components of PDC.
From the discussion part of this paper, the authors revealed that in other related studies on PDC and its constituent E2 component of bovine kidney, “limited proteolysis by leupeptin-sensitive enzyme (termed “inactivase”) and papain have obtained the similar results as done by trypsin”(9,26). However, though these proteases have no effect on any component activities of PDC during the whole inactivation reaction and the three component enzymes can retain their complete enzymatic activity, in contrast to trypsin, they may lack of high specificity to cleave the targeted enzymes. In addition, when treated with the inactivase or papain, E1 and E3 of PDC are not cleaved. Furthermore, “the inactivase is not easy to purify and can be obtained only in small quantities compared to other proteases” (9). This in turn reflects that the authors of this paper chose a more appropriate protease to conduct the limited proteolysis of PDC of bovine heart to facilitate the analysis of cleaved products.
Morphologic comparison of trypsin-modified transacetylase and unmodified transacetylase
The authors compared the morphologic appearance of both trypsin-modified transacetylase and unmodified transacetylase in electron micrographs illustrated in Figure 3A and 3B, respectively. They claimed that the difference of diameter between these two components can be attributed to an interpretative model showed in Figure 3C. Actually, I can hardly agree with the authors’ hypothesis because though they proposed that the modified fragment, i.e. the subunit-binding domain contributes to the transacetylase’s quaternary structure, there is no direct evidence to support this model’s constitution. Furthermore, the authors also did ultracentrifugation experiments to demonstrate that limited tryptic digestion of transacetylase would prevent the reassemblage of it and other two components (E1 and E3) of PDC. Combined with the previous research progress, the subunit structure of PDC of bovine heart is quite different from that of E.coli in many aspects. Take E2 of PDC as an example. Previous study has revealed that “E2 of bovine heart consists of 60 apparently identical polypeptide chains arranged with icosahedral point group symmetry” (4) whereas “in E. coli the number of polypeptide chains arranged with octahedral symmetry is 24” (10, 11). “Moreover, the transacetylase subunit of E. coli consists of two different domains: a subunit-binding domain and a lipoyl domain containing two lipoyl moieties” (12, 13) whereas “the lipoyl domain and the intact transacetylase subunit of bovine heart contain only one lipoyl moiety” (5). Last, the subunit-binding domain of transacetylase from E.coli contains the binding sites for other two enzymes (E1 and E3) of PDC. But in bovine heart E2, the subunit-binding domain cannot interact with these two enzymes without the presence of the lipoyl domain.
Purification and Separation of Tryptic Fragments
There are two different approaches to separate the trypsin-modified transacetylase (assemblage of subunit-binding domains) from the lipoyl domain: ultracentrifugation and gel filtration. The latter one is preferential due to the difficulty to redissolve the modified transacetylase by ultracentrifugation. Figure 4 showed a typical elution profile of gel filtration on sepharose. According to the principles of gel filtration, the modified transacetylase will be eluted earlier than the lipoyl domain due to the size difference. The authors concluded from the profile’s two major peaks that the catalytic site for transacetylation was not affected by limited tryptic digestion. On the other hand, Figure 5 indicated the gel patterns of purified subunit-binding domain and lipoyl domain by SDS-PAGE. As the molecular weight of the subunit-binding domain is smaller than that of the lipoyl domain, its stained gel band moves faster.
Different Features of Lipoyl Domain and Subunit-binding Domain
According to the data from Tableâ… , for the lipoyl domain, its molecular weight of 28.6kDa determined by sedimentation equilibrium analysis is quite lower than that estimated by SDS-PAGE (37.0kDa). The authors provided with several other physical parameters to indicate that the lipoyl domain possesses an extended structure and by estimating the approximate isoelectric point of the lipoyl domain, they claimed that its acidic nature may in part contribute to its anomalous migration on SDS gels. However, they did not conduct any experiments or supply data to confirm their viewpoint. I personally think that since the lipoyl domain contain a large proportion of acidic Glu residues, this perhaps will have effect on electrophoresis as it can be influenced by aberrant charges of molecules.
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In summary, the results of this paper indicate that the dihydrolipoyl transacetylase (E2) subunit of pyruvate dehydrogenase complex (PDC) from bovine heart is made up of two different fragments termed the subunit-binding domain and the lipoyl domain. The former (Mr≈26,000) possesses compact structure and its assemblage confers the morphologic appearance of transacetylase in electron microscope. The latter (Mr≈28,600), however, which contains a covalently bound lipoyl moiety and hence exhibits anomalous migration in SDS-PAGE, is bound to the subunit-binding domain with an extended structure. The authors used limited trypsin digestion and isotope labeling to successfully obtain and identify these two fragments. Followed by proteolysis of E2, they then conducted a serious of experiments to analyze and purify the digestion products from PDC and E2. Nonetheless, there are several missing controls which make their results less convincing and similarly when they explore the morphology of transacetylase and the anomalous migration of the lipoyl domain, the authors did not provide sufficient evidence to support their perspectives.
Finally, this paper’s achievements have influenced correlated research fields on many aspects. There are incredibly increasing studies that focus on not only the “molecular biology and biochemistry of PDC” (9) but also on “PDC from other organisms such as yeast” (10). All these amazing progress again prove the importance of PDC and its component enzymes which serve as a hinge to link the most extensive biological metabolism processes in organisms.
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