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PubMed2026

Auxin-induced ARF transcription factor degradation defines tissue boundaries.

How organs partition themselves into discrete domains with distinct functions is a fundamental question in biology. The gynoecium of flowering plants provides an excellent system to address this question. Here, we show that the boundary between the stigma and style at the gynoecium apex is established by the complementary distribution of the phytohormone auxin and the Auxin Response Factor (ARF), ETTIN (ETT). Mechanistically, auxin induces ETT protein destabilization via the ubiquitin-proteasome pathway. A short sequence motif within an intrinsically disordered region is required for this auxin-triggered degradation. Disruption of this motif leads to ectopic ETT accumulation at the gynoecium apex and consequently abolishes stigma-style boundary development. We further demonstrate that this previously unrecognized mode of auxin-induced ARF instability is evolutionarily conserved among ETT orthologs across angiosperms. In summary, this study reveals how graded auxin distribution affects ARF transcription factor activity, contributing to the establishment of the stigma-style boundary, ensuring correct gynoecium formation and reproductive success in flowering plants.

Indoleacetic Acids↗

PubMed1978

Newer aspects of pernicious anemia.

Although readily treatable with vitamin B12, pernicious anemia continues to captivate investigative endeavors of those interested in the pathophysiology and pathogenesis of this disorder. Notable advances have been made in understanding properties of intrinsic factor, vitamin B12-binding proteins, structure and de novo synthesis of vitamin B12, mechanism of action of vitamin B12-dependent enzymes in man, and metabolic consequences of reduced activities of these enzymes in pernicious anemia. Similarly, newer morphological observations have given information regarding pathogenesis of some of the cytological abnormalities found in megaloblasts, and recent cytochemical studies have shed light on abnormalities of nuclear and cytoplasmic constituents in vitamin B12-deficient cells. Both cellular and humoral factors may contribute to immune-mediated processes in pernicious anemia, although as yet, it has not been established with certainty that pernicious anemia is an autoimmune disorder. As we look ahead, it will be important to define the process or processes responsible for atrophic gastritis, which is the pathophysiological basis of pernicious anemia. Likewise, advances in biophysics used in the study of cell membranes, cell surface phenomena, and metallic ion transport may find applicability in the study of pernicious anemia and perhaps provide further insights into metabolic abnormalities responsible for the development of megaloblastosis.

Anemia, Pernicious↗

PubMed2026

The pseudokinase domain PK1 of UNC-89/obscurin is required for mitochondrial morphology and function in C. elegans.

UNC-89 is a giant modular protein located at the sarcomeric M-line of C. elegans striated muscle and is required for sarcomere organization and function. UNC-89 contains two protein kinase domains, PK1 and PK2, separated by 850 residues, that includes a 645-residue long intrinsically disordered sequence that acts like an elastic spring. Bioinformatic analysis suggests that PK2 is an active kinase whereas PK1 is a pseudokinase. We recently reported that a genome-edited worm, unc-89(sf22), that expresses UNC-89 carrying a kinase-inactivating point mutation in PK2 has an unusual phenotype with normally organized sarcomeres and SR, normal muscle function and yet fragmented mitochondria, increased ATP levels, increased glycolysis and alterations in electron transport chain complexes and respiration. Here, we show that a genome-edited worm unc-89(sf23), that expresses UNC-89 with an in-frame deletion of the C-lobe of PK1 has approximately the same phenotype as the PK2 catalytically dead mutant. The fact that mutations in two different regions of UNC-89 result in a mitochondrial phenotype is further evidence of communication between the sarcomere and mitochondria. We further demonstrate that in vitro PK2 interacts with full length PK1 and the C-lobe of PK1. The protein kinase domains of giant sarcomeric proteins are autoinhibited by parts of their own sequence, and this is also likely for PK2, but the mechanism by which PK2 would be activated is unknown. Our data is compatible with a model in which PK1 interacts with PK2 and thereby stimulates PK2 kinase activity.

Animals↗

PubMed2025

BAV-LLPS: a database of bacterial, archaea, and virus liquid-liquid phase separation proteins.

MOTIVATION: Liquid-liquid phase separation (LLPS) is a key process underlying the formation of biomolecular condensates, such as membrane-less organelles, that compartmentalize biochemical processes inside the cells. While LLPS has been extensively studied in eukaryotes, its role in bacteria, archaea, and viruses remains far less characterized. Recent studies in bacteria have revealed that LLPS-driven condensates play critical roles in RNA processing, stress response, and pathogenicity. Similarly, many viruses exploit LLPS to facilitate crucial steps in their infection cycles, including viral entry, genome replication, assembly, and host immune evasion. RESULTS: In this work, we introduce a hand-curated database of LLPS proteins from bacteria, archaea, and viruses (BAV-LLPS Database). This resource, extended through sequence similarity searches, comprises over 5000 proteins and integrates diverse data including biological annotations, sequence features, predicted disordered regions, LLPS per site probability, and AlphaFold2-based structural models. Additionally, our web server enables users to explore both the curated and homologous derived datasets, providing a platform to uncover evolutionary relationships and intrinsic and differential properties of LLPS proteins across various taxonomic groups. This work seeks to deepen our understanding of LLPS mechanisms beyond eukaryotic organisms, emphasizing their significance across diverse life forms. It also aims to foster the development of specialized predictive tools that will facilitate the exploration and characterization of LLPS processes in a wide array of living organisms, thereby contributing to advancements in both fundamental biological research and applied biomedical sciences. AVAILABILITY AND IMPLEMENTATION: BAV-LLPS DB is freely accessible at https://bav-llps-db.bioinformatica.org/. The data can be retrieved from the website. The source code of the database can be downloaded from https://bav-llps-db.bioinformatica.org/download.

Databases, Protein↗

PubMed2026

Systematic identification of germ granule proteins reveals specialized roles in RNAi and small RNA inheritance.

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. However, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has coevolved with PRG-1 across clade V nematodes, with a conserved structured N terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily coadapted PIWI interactor critical for siRNA biogenesis.

Animals↗

PubMed2026

Small GTPase RAN-driven PNET2 oligomerization and phase separation at the nuclear lamina promote nuclear envelope integrity in plants.

The nuclear envelope is a fundamental organizer of eukaryotic cells, yet how plants regulate its architecture and integrity remains poorly understood. In this study, we identified the plant inner nuclear membrane protein PLANT NUCLEAR ENVELOPE TRANSMEMBRANE 2 (PNET2) as a scaffold that maintains nuclear envelope integrity and genome stability. Loss of PNET2 function compromises nuclear membrane structure and sensitizes cells to DNA damage, whereas overexpression drives aberrant nuclear membrane expansion. Biochemically, PNET2 cooperates with the nuclear lamin protein KAKU4 and CROWDED NUCLEI 1 within the nuclear lamina to promote nuclear membrane remodeling, a process driven by biomolecular condensate formation via their intrinsically disordered regions. We further uncovered a direct interaction between PNET2 and the small GTPase RAN. Structural modeling and biochemical analyses revealed that its active GTP-bound form stimulates PNET2 oligomerization, potentially promoting its phase separation to drive membrane expansion. Genetic analyses showed that PNET2 and RAN function in a shared pathway essential for nuclear membrane integrity. Together, our findings define a regulatory module that orchestrates GTPase signaling to sustain nuclear membrane homeostasis in plants, positioning PNET2 as a nexus linking membrane dynamics, nuclear lamina organization, and genome protection.

PNET2↗

PubMed2026

ATRX Condensates as Candidate Organizers of Enhancer-Centered Nuclear Microenvironments in Neural Progenitors: A Hypothesis for Enhancer-Associated ATRX Function in Neural Progenitors.

Neural progenitor cells (NPCs) must preserve lineage identity while remaining responsive to developmental cues. Here, we discuss the hypothesis that ATRX condensates help organize enhancer-centered nuclear microenvironments in NPCs. ATRX has long been studied in heterochromatin maintenance, histone variant deposition, and chromatin remodeling; earlier work has also shown that ATRX can occupy euchromatic and active regulatory regions and contribute to transcriptional regulation. Recent evidence in human NPCs indicates that ATRX forms nuclear puncta with condensate-like properties, associates with neurogenic enhancer-rich regions, and incorporates regulatory factors such as CHD7 and p300. Perturbation of ATRX condensate formation is associated with changes in enhancer-associated ATRX occupancy, neural gene-expression programs, and neuroepithelial organization, suggesting a regulatory mode that may complement canonical heterochromatin-associated functions. We propose a dual-mode model in which folded domains contribute to chromatin anchoring at repressive regions, whereas intrinsically disordered regions support condensate-associated organization at active developmental enhancers. We emphasize that whether ATRX condensates activate enhancers de novo, stabilize pre-existing enhancer states, buffer transcriptional variability, or primarily organize cofactor localization remains unresolved. We also discuss limitations of the current evidence and outline acute, locus-specific experiments needed to test the model.

X-linked Nuclear Protein↗

PubMed1978

Electron microscopy in the early diagnosis of genetic disorders of the skin.

Electron microscopy is shown to represent an effective tool in the early diagnosis of genetic disorders. On the basis of ultrastructural findings in various dominant and recessive types of ichthyoses and epidermolyses, defects of structural proteins of the skin proved to form the main intrinsic pathogenetic feature of some dominantly inherited types whereas quantitative impairments characterize their closely resembling recessive counterparts. Most of the diseases concerned severely disable the involved patients. Early diagnosis is therefore of high importance. In those cases where an exact diagnosis in newborn children is difficult or impossible with respect to their clinical features, electron microscopy provides the clinician with reliable and significant criteria to differentiate between closely resembling entities.

Epidermolysis Bullosa↗

PubMed2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32).

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the system's three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds (~1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule Förster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

Journal Article↗

PubMed1978

Peripheral amino acid levels in patients with cancer.

Peripheral arterial and venous whole blood amino acid concentrations were determined in four groups of subjects after an overnight fast 1) normal people, 2) patients with cancer who had not lost body weight, 3) subjects with cancer who had lost more than 20% of body weight and 4) patients who had lost more than 20% of body weight from diminished intake due to cause other than cancer. Comparison of the arterial blood levels in the four groups showed that patients with cancer and weight loss had amino acid patterns different from patients who were malnourished for other reasons. Branched chain amino acids were normal in patients with malignant disease. Some gluconeogenic amino acids were reduced as in other subjects with weight loss but the characteristic rise in glycine seen with malnutrition was not present. Arterio venous differences in whole blood across the forearm showed no evidence of increase in venous excess in patients with progressive malignant disease, indicating no excessive protein catabolism in muscle tissue. The data are consistent with increased gluconeogenesis in malnourished cancer subjects, probably due to intrinsic change in hepatic metabolism.

Amino Acids↗

PubMed2026

mRNA therapy: A novel approach for retinal neurodegenerative diseases.

Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.

administration↗

PubMed1976

Neutrophil function in gram-negative rod bacteremia. The interaction between phagocytic cells, infecting organisms, and humoral factors.

To assess the phagocytic and bactericidal function of neutrophils in the acute stages of gram-negative rod bacteremia, cells from 30 nonleukopenic patients were studied in a test system utilizing plasma obtained simultaneously with culture-positive blood, the autologous infecting strain, and two laboratory test strains of Staphylococcus aureus and Pseudomonas aeruginosa. Results were compared to those obtained with normal neutrophils and plasma. Patient and control plasma were simultaneously tested with each source of phagocytic cells to localize any abnormalities. Four patients had a defect against their infecting strain, 33% of the inoculum phagocytized and killed versus 80% by controls. In these cases differences were localized to the patients' plasma, as normal plasma tested with patients' cells reversed the defect. Thus, four patients had impaired opsonization when compared to normal controls, but we also observed that 11 of 30 bacteremic isolates, all Escherichia coli, showed absolute or relative resistance to phagocytosis in the patient and control assay system. No intrinsic granulocyte killing abnormalities were noted. There was poor correlation between results obtained with infecting strains compared to laboratory test organisms. We conclude that in patients without evidence of an inherited neutrophil bactericidal disorder, recurrent infection, or treatment with cytotoxic drugs, intrinsic bactericidal defects are uncommon at the onset of gram-negative bacteremia, and impaired opsonization is the most commonly encountered cause of neutrophil dysfunction.

Blood Bactericidal Activity↗

PubMed1976

Hemolytic anemias. Failure of the red cell membrane.

The normal erythrocyte membrane is composed of nearly equivalent amounts of lipid and protein. The lipid portion of the membrane has been well studied. Even though de novo synthesis of lipid does not occur in human red cells, many biochemical pathways exist which facilitate detoxification of lipid breakdown products and lipid renewal. Rare defects in these processes are associated with hemolytic disorders. Recent studies have revealed that the membrane proteins are diverse and suggest that protein dysfunction may also account for clinical disease. Protein and lipid are entwined in a physicochemical relationship which is probably best depicted by the classic lipid bilayer with interspersed proteins in both the inner and outer surfaces and also spanning the bilayer. Membrane failure results in hemolytic anemia. This failure can be intrinsic, caused by abnormal lipid or protein constituents; or extrinsic, with a normal membrane being unable to counteract physical, chemical or immunologic stress. Clinical examples of membrane failure and hemolytic anemia can be separated into three groups according to the predominant mechanism of the hemolysis: fragmentation, whole-cell lysis, and filtration and entrapment. Although these mechanisms can act separately or in concert, the final hemolytic destruction of the cell can usually be traced to a failure of membrane function.

Abetalipoproteinemia↗

PubMed1977

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.

Amino Acid Sequence↗

PubMed1979

Clinical correlations between long-term (IgE) and short-term (IgG S-TS) anaphylactic antibodies in atopic and 'non-atopic' subjects with respiratory allergic disease.

The sera of atopic and non-atopic persons with allergic pulmonary disorders were examined for long-term sensitizing, IgE and short-term sensitizing heat-stable (S-TS) antibodies which were present separately or together in the sera of some patients sensitive to antigens such as budgerigar serum proteins and Aspergillus funigatus. In fourteen atopic patients with extrinsic asthma, six had both types of antibody to common allergens, and of nine non-atopic patients with crytogenic (intrinsic) asthma, four had only heat-stable short-term sensitizing antibodies. The sera of atopic subjects with type I prick test reactions and positive RAST's, showed specific IgE antibody by baboon PCA tests to budgerigar serum proteins, A. funigatus, Timothy grass pollen extract and hen egg extract, and not to Dermatophagoides farinae, possibly because of naturally occurring mite antibodies in the baboon. The sera of non-atopic asthmatics, who had given negative prick test but positive immediate, dual or late intracutaneous tests, and only late asthmatic reactions, contained precipitins in most cases and gave little or no RAST reaction. On baboon PCA these sera contained either, S-TS antibody alone, or S-TS plus long-term sensitizing antibody, or long-term sensitizing antibody alone. Some of the sera with long-term sensitizing antibody contained blocking antibody which could diffuse away in the 24 hr delay for the baboon PCA test and could also be responsible for the negative RAST. Tests with insoluble anti-IgE immuno-adsorbents on two sera from persons sensitive to aspergillus confirmed that the S-TS activity was not due to IgE, and on two sera with negative RAST and negative prick tests to budgerigar serum antigens confirmed that the 24 hr monkey PCA responses were due to IgE.

Allergens↗

PubMed2026

The piRNA pathway mediates transcriptional silencing of LTR retrotransposons in ovaries and somatic tissues of Aedes mosquitoes.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by suppressing transposable elements in animal germlines. Despite its well-established function in the animal germline, piRNAs and PIWI proteins are expressed in somatic tissues across arthropod species, and their functions outside the gonads remain poorly understood. Aedes albopictus mosquitoes express four PIWI genes, Piwi4, Piwi5, Piwi6, and Ago3, in both gonadal and somatic tissues. Here, we generated Piwi6 knockout (KO) Ae. albopictus cell lines and observed a substantial upregulation of long terminal repeat retrotransposons, including a full-length endogenous retrovirus that we named Aedes albopictus Endogenous Retrovirus-1 (AalERV1). Nascent RNA sequencing and Cleavage Under Targets and Tagmentation (CUT&Tag) analyses revealed that Piwi6 silences AalERV1 transcriptionally by guiding the deposition of the repressive H3K9me3 histone mark. Consistently, Piwi6 localized to both the cytoplasm and nucleus, with sequences in the intrinsically disordered region guiding nuclear translocation. Reintroduction of full-length GFP-Piwi6, but not a mutant GFP-Piwi6 defective in nuclear localization, rescued AalERV1 repression in Piwi6 KO cells. Importantly, Piwi6-mediated control of AalERV1 was recapitulated in vivo as Piwi6 knockdown increased AalERV1 expression in both ovaries and somatic tissues of Ae. albopictus mosquitoes. These results establish Aedes mosquitoes as a model to study nuclear PIWI functions and suggest that somatic piRNA-mediated transposon silencing is evolutionarily conserved across arthropod species.

Animals↗

PubMed1977

Participation of Hageman factor dependent pathways in human disease states.

Abnormalities of Hageman factor dependent pathways have been described in a wide variety of human disease states. Congenital deficiencies of factor XII (Hageman trait) prekallikrein (Fletcher trait) and high molecular weight kininogen (Williams, Fitzgerald and Flaujeac traits) although resulting in profound in vitro changes, do not cause in vivo difficulties. In contrast, deficiency of C1 esterase inhibitor (hereditary angioedema) results in significant morbidity and mortality. Acquired diseases may exhibit decreased synthesis of these three proteins in cirrhosis and dengue fever. In vivo activation of factor XII initiated pathways occur in septic shock, disseminated or localized intravascular coagulation, typhoid fever, polycythemia vera, hyperbetalipoproteinemia, coronary artery disease, nephrotic syndrome, transfusion reactions, hemodialysis and extracorporeal bypass. Activation of both the intrinsic system and tissue mediators contribute to the vasomotor phenomena in carcinoid syndrome and postgastrectomy dumping. Roles for factor XII, prekallikrein and kininogen have been suggested in gouty arthritis, allergic disorders and cystic fibrosis but the evidence is not yet convincing in these disorders.

Angioedema↗

PubMed1976

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.

Amino Acid Sequence↗