Formation Of High Molecular Weight Complexes Research Articles

Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain.

Integral membrane proteins play acentral role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3×FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexesin additionto formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and theirprotein complexes.

0521 Chemical characteristics and enhanced hepatoprotective activities of Maillard-reaction products derived from milk protein-sugar system

The objective of this study was to investigate the characteristics, antioxidative properties, and hepatoprotective effects of Maillard reaction products (MRP) from milk protein reacted with sugars. The MRP were obtained from milk protein, whey protein concentrates and sodium caseinate, using 2 types of sugars, lactose and glucose, by heating the mixture at 55°C for 7 d in a sodium phosphate buffer (pH 7.4). Changes in the chemical modification of the milk protein were monitored by measuring the protein-bound carbonyls and PAGE protein profiles. The results showed that the amount of protein-bound carbonyls increased after Maillard reaction (MR). In addition, sodium dodecyl sulfate-PAGE analysis indicated a formation of high-molecular weight complexes through MR. The modification sites induced by MR of milk protein were monitored by matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis of tryptic-digested gel spots of MRP. As a result, modification and their localization in AA sequence of MRP was identified. Also, the MRP showed higher antioxidant activities than the intact milk protein, and they reduced intracellular reactive oxygen species production and inhibited the depletion of the reduced glutathione concentrations in the HepG2 cells. In particular, glucose-sodium caseinate MRP showed the highest biological activities among all MRP. Therefore, these results suggest that the MRP from milk protein reacting with sugars possess effective antioxidant activity and have a protective ability against oxidative damage.

Chemical characteristics and enhanced hepatoprotective activities of Maillard reaction products derived from milk protein-sugar system

The objective of this study was to investigate the characteristics, antioxidative properties, and hepatoprotective effects of Maillard reaction products (MRP) from milk protein reacted with sugars. The MRP were obtained from milk protein, whey protein concentrates and sodium caseinate, using 2 types of sugars, lactose and glucose, by heating the mixture at 55°C for 7d in a sodium phosphate buffer (pH 7.4). Changes in the chemical modification of the milk protein were monitored by measuring the protein-bound carbonyls and PAGE protein profiles. The results showed that the amount of protein-bound carbonyls increased after Maillard reaction (MR). In addition, sodium dodecyl sulfate-PAGE analysis indicated a formation of high-molecular weight complexes through MR. The modification sites induced by MR of milk protein were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of tryptic-digested gel spots of MRP. As a result, modification and their localization in AA sequence of MRP was identified. Also, the MRP showed higher antioxidant activities than the intact milk protein, and they reduced intracellular reactive oxygen species production and inhibited the depletion of the reduced glutathione concentrations in the HepG2 cells. In particular, glucose-sodium caseinate MRP showed the highest biological activities among all MRP. Therefore, these results suggest that the MRP from milk protein reacting with sugars possess effective antioxidant activity and have a protective ability against oxidative damage.

Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein.

Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr(701) by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/β. Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr(701) residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr(701), further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/β pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone

Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone.

The RNA Chaperone and Protein Chaperone Activity of Arabidopsis Glycine-Rich RNA-Binding Protein 4 and 7 is Determined by the Propensity for the Formation of High Molecular Weight Complexes

RNA chaperones and protein chaperones are cellular proteins that can aid the correct folding of target RNAs and proteins, respectively. Although many proteins possessing RNA chaperone or protein chaperone activity have been demonstrated in diverse organisms, report evaluating the RNA chaperone and protein chaperone activity of a given protein is severely limited. Here, two glycine-rich RNA-binding proteins in Arabidopsis thaliana (AtGRPs), AtGRP7 exhibiting RNA chaperone activity and AtGRP4 exhibiting no RNA chaperone activity, were investigated for their protein chaperone activity. The heat-induced thermal aggregation of a substrate protein was significantly decreased with the addition of AtGRP4 depending on protein concentration, whereas the thermal aggregation of a substrate protein was further increased with the addition of AtGRP7, demonstrating that AtGRP4 but not AtGRP7 possesses protein chaperone activity. Size exclusion chromatography and electron microscopy analyses revealed that the formation of high molecular weight (HMW) complexes is closely related to the protein chaperone activity of AtGRP4. Importantly, the additional 25 amino acids at the N-terminus of AtGRP4 are crucial for HMW complex formation and protein chaperone activity. Taken together, these results show that the formation of HMW complexes is important for determining the RNA chaperone and protein chaperone activity of AtGRP4 and AtGRP7.

TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells

IntroductionElevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques. Materials and methodsThe effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy. ResultsTM5275 at concentrations of 20 and 100μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells. ConclusionsThe profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.

SDS-PAGE Analysis of Soluble Proteins in Reconstituted Milk Exposed to Different Heat Treatments

This paper deals with the investigation of the impact of the heat treatment of reconstituted skim milk conducted at different temperatures, and the adding of demineralized whey on the protein solubility, soluble protein composition and interactions involved between proteins in a chemical complex. Commercial skim milk has been reconstituted and heat treated at 75°C, 85°C and 90°C for 20 minutes. Demineralized whey has been added in concentrations of 0.5%, 1.0 and 2.0%. The soluble protein composition has been determined by the polyacrilamide gel electrophoresis (SDS-PAGE) and by the densitometric analysis. Due to the different changes occurred during treatments at different temperatures, proteins of heat-treated samples containing added demineralized whey have had significantly different solubility. At lower temperatures (75°C and 85°C) the adding of demineralized whey decreased the protein solubility by 5.28%-26.41%, while the addition of demineralized whey performed at 90°C increased the soluble protein content by 5.61%- 28.89%. Heat treatments, as well as the addition of demineralized whey, have induced high molecular weight complex formation. β-Lg, α-La and κ-casein are involved in high molecular weight complexes. The disulfide interactions between denatured molecules of these proteins are mostly responsible for the formation of coaggregates. The level of their interactions and the soluble protein composition are determined by the degree of temperature.

Targeting the Oligomerization Domain of ETO Interferes with RUNX1/ETO Oncogenic Activity in t(8;21)-Positive Leukemic Cells

About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21), which generates the oncogenic fusion protein RUNX1/ETO. RUNX1/ETO has a modular structure and contains several docking sites for heterologous proteins, including transcriptional co-repressors like N-CoR, SMART, and mSIN3A. RUNX1/ETO is found in high molecular weight complexes, which are crucial for the block in myeloid differentiation observed in RUNX1/ETO-transformed cells. Essential for high molecular weight complex formation is the nervy homology region 2 (NHR2) within ETO, which serves as interacting surface for oligomerization as well as association with members of the ETO protein family. Here, we show that the expression of a fusion peptide consisting of 128 amino acids (NC128), including the entire NHR2 domain of ETO, disrupts the stability of the RUNX1/ETO high molecular weight complexes, restores transcription of RUNX1/ETO target genes, and reverts the differentiation block induced by RUNX1/ETO in myeloid cells. In the presence of NC128, RUNX1/ETO-transformed cells lose their progenitor cell characteristics, are arrested in cell cycle progression, and undergo cell death. Our results indicate that selective interference with the oligomerization domain of ETO could provide a promising strategy to inhibit the oncogenic properties of the leukemia-associated fusion protein RUNX1/ETO.

The Mechanism of γ-Secretase Activities through High Molecular Weight Complex Formation of Presenilins Is Conserved inDrosophila melanogaster and Mammals

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Abeta42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Abeta1-42 from human betaAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals.

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.

Sendai virus C protein physically associates with Stat1.

The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined. Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN-alpha/beta and IFN-gamma signalling. We found that both unphosphorylated Stat1 and tyrosine-phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low-salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1-HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY-Stat1 homodimer to pY-Stat1-HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high-salt buffer, and under this stringent (high-salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY-Stat1 were co-precipitated with anti-C antibody. The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.

Multiple Regions of ETO Cooperate in Transcriptional Repression

In acute myeloid leukemias (AMLs) with t(8;21), the transcription factor AML1 is juxtaposed to the zinc finger nuclear protein ETO (Eight-Twenty-One), resulting in transcriptional repression of AML1 target genes. ETO has been shown to interact with corepressors, such as N-CoR and mSin3A to form complexes containing histone deacetylases. To define regions of ETO required for maximal repressor activity, we analyzed amino-terminal deletions in a transcriptional repression assay. We found that ETO mutants lacking the first 236 amino acids were not affected in their repressor activity, whereas a further deletion of 85 amino acids drastically reduced repressor function and high molecular weight complex formation. This latter mutant can still homodimerize and bind to N-CoR but shows only weak binding to mSin3A. Furthermore, we could show that a "core repressor domain" comprising nervy homology region 2 and its amino- and carboxyl-terminal flanking sequences recruits mSin3A and induces transcriptional repression. These results suggest that mSin3A and N-CoR bind to ETO independently and that both binding sites cooperate to maximize ETO-mediated transcriptional repression. Thus, ETO has a modular structure, and the interaction between the individual elements is essential for the formation of a stable repressor complex and efficient transcriptional repression.

Soluble thrombomodulin quenches thrombin-mediated neutralization of PAI-1 activity and inhibits fibrinolysis through a TAFI independent mechanism

Objective: To evaluate the effect of thrombomodulin on thrombin-mediated neutralization of plasminogen activator inhibitor 1 (PAl-1) activity which results in the enhancement of the fibrinolytic activity. Design: We studied the effect of recombinant human soluble TM (rhs TM) on the interaction between human thrombin and PAI-1. Its subsequent effect on tissue plasminogen activator (tPA)-induced lysis of PAl-1 enriched fibrin clot was also evaluated. Results: rhsTM abolished the high molecular weight complex formation between thrombin and PAl-1 and quenched the neutralization of PAl-1 activity by thrombin in a dose-dependent manner. rhsTM also caused dose-dependent inhibition of tPA-induced lysis of PAl-1 enriched fibrin clots in a purified system, which had been shown to be accelerated by increasing concentration of thrombin by neutralizing PAl-1 activity. This inhibition was not observed when PAl-1 was not present in the fibrin clot. Euglobulin clot lysis time (ECLT), which is determined by the balance between tPA and PAl-1, was prolonged by rhsTM. This prolongation was partially abolished by anti-PAl-1 polyclonal IgG, but was unaffected by potato carboxyl peptidase inhibitor. Conclusion: The inhibition of thrombin-dependent enhancement of fibrinolysis by TM appears to involve a mechanism of quenching of thrombin-mediated neutralization of PAl-1 activity which is independent of thrombin activatable fibrinolysis inhibitor (TAFl).

117 Impact of adipose tissue on plasma plasminogen activator inhibitor-1 in dieting obese women

Objective: To evaluate the effect of thrombomodulin on thrombin-mediated neutralization of plasminogen activator inhibitor 1 (PAl-1) activity which results in the enhancement of the fibrinolytic activity.Design: We studied the effect of recombinant human soluble TM (rhs TM) on the interaction between human thrombin and PAI-1. Its subsequent effect on tissue plasminogen activator (tPA)-induced lysis of PAl-1 enriched fibrin clot was also evaluated.Results: rhsTM abolished the high molecular weight complex formation between thrombin and PAl-1 and quenched the neutralization of PAl-1 activity by thrombin in a dose-dependent manner. rhsTM also caused dose-dependent inhibition of tPA-induced lysis of PAl-1 enriched fibrin clots in a purified system, which had been shown to be accelerated by increasing concentration of thrombin by neutralizing PAl-1 activity. This inhibition was not observed when PAl-1 was not present in the fibrin clot. Euglobulin clot lysis time (ECLT), which is determined by the balance between tPA and PAl-1, was prolonged by rhsTM. This prolongation was partially abolished by anti-PAl-1 polyclonal IgG, but was unaffected by potato carboxyl peptidase inhibitor.Conclusion: The inhibition of thrombin-dependent enhancement of fibrinolysis by TM appears to involve a mechanism of quenching of thrombin-mediated neutralization of PAl-1 activity which is independent of thrombin activatable fibrinolysis inhibitor (TAFl).

Characterization of precursor and secreted forms of human angiotensinogen.

To define the basis of the heterogeneity of angiotensinogen, we have characterized the immunoreactivity of high molecular weight (HMW) and low molecular weight (LMW) plasma angiotensinogen, the angiotensinogen precursor synthesized by cell-free translation, and angiotensinogen secreted by human hepatoma (Hep G2) cells. Angiotensinogen precursor synthesized by rabbit reticulocyte lysate primed with RNA prepared from liver or Hep G2 cells was compared with angiotensinogen secreted by Hep G2 cells by using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). So as to assess the contribution of N-glycosylation of angiotensinogen, Hep G2 cells were incubated in the presence of tunicamycin. Glycosylation of secreted angiotensinogen was further characterized by using chromatography on concanavalin A-Sepharose, digestion with neuraminidase, and treatment with trifluoromethane sulfonic acid. In Sephadex G-200 column chromatography, HMW plasma angiotensinogen eluted just after the column void volume and was clearly separated from LMW angiotensinogen which eluted just before bovine serum albumin. Both HMW and LMW plasma angiotensinogen were shown to bind to monoclonal and polyclonal antibodies raised against pure LMW angiotensinogen. Only one angiotensinogen precursor (mol wt 50,000) was identified by cell-free translation which, after cleavage by renin, was reduced to mol wt 45,600. Angiotensinogen secreted by Hep G2 cells showed electrophoretic heterogeneity (mol wt 53,100-65,400). Tunicamycin-treated Hep G2 cells secreted five discrete forms of angiotensinogen, a predominant form of mol wt 46,200, with other forms (mol wt 46,800, 48,100, 49,200, and 49,600) representing 10% of secreted angiotensinogen. All five forms showed a similar reduction in molecular weight after cleavage by renin. The predominant 46,200-mol wt protein represented nonglycosylated angiotensinogen in that, after cleavage by renin, it had an electrophoretic mobility (mol wt 45,600) identical to the desangiotensin I-angiotensinogen resulting from renin cleavage of the angiotensinogen precursor. The other higher molecular weight forms of angiotensinogen secreted by tunicamycin-treated Hep G2 cells were shown to represent O-glycosylated angiotensinogen in that they were reduced to 46,200 mol wt by treatment with trifluoromethane sulfonic acid. Dexamethasone (10(-7) and 10(-6)M) stimulated angiotensinogen secretion by Hep G2 cells two- to fourfold, both in the absence and presence of tunicamycin. However, a small stimulatory effect of mestranol (10(-7) M) was evident only in the presence of tunicamycin. Neither dexamethasone nor mestranol influenced the electrophoretic pattern (SDS-PAGE) of angiotensinogen secreted by Hep G2 cells. However, when incubation media were chromatographed on Sephadex G-200 with subsequent immunoprecipitation of the column fractions, both dexamethasone and mestranol were shown to stimulate the secretion of HMW angiotensinogen (eluting just after the column void volume) which, on SDS-PAGE, migrated in a position identical to LMW angiotensinogen. From these studies, we conclude that all forms of human angiotensinogen are derived from a single precursor. The heterogeneity of secreted angiotensinogen represents differences in posttranslational processing of angiotensinogen. This processing includes both N- and O-glycosylation, and also the formation of HMW complexes (HMW angiotensinogen) through association either with other angiotensinogen molecules or with some other protein(s) whose secretion by hepatocytes is stimulated by glucocorticoids and estrogens.

High molecular weight complex formation of rat liver lysyl-tRNA synthetase reduces enzyme lability to thermal inactivation

Eukaryotic aminoacyl-tRNA synthetases tend to occur in high molecular weight multienzyme complexes in contrast to the prokaryotic counterparts. The functional significance of complex formation is not understood but stabilization of enzyme activities in the complex(es) has been suggested as one of many possible functions. We provide evidence that the 24S complex lysyl-tRNA synthetase activity is more resistant to thermal inactivation than the 6S free lysyl-tRNA synthetase activity at 30°, 37°, and 43°C.