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1. Sequence analysis of the NADPH domain (residues 158 - 293) and of the interface domain (365 - 478) was based on 12 CNBr fragments, which were isolated using ion-exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid-phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. 2. In the NADPH domain, residue 197, which is involved in an induced-fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chainfold in other proteins. 3. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallographic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, Ile29, Leu34, Tyr13, Phe14, Lys34. His16, Arg17, and Trp3. From these data an mr of 2 × 51 600 was calculated for the FAD-free apoenzyme and an Mr of 2 × 52400 for the holoenzyme. [ABSTRACT FROM AUTHOR]

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

The ability ofEntamoeba histolyticaTATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study thein vitroEhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box forE. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found inE. histolyticagene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (±0.39) × 10−11 and 1.60 (±0.37) × 10−10 m. TATTTAAA and TAT_ _AAA motifs presented the lowestKD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding. [ABSTRACT FROM AUTHOR]

Histopathological hallmarks of Alzheimer's disease (AD) are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the aggregated amyloid‐beta peptide along with metal ions (copper, iron or zinc). In addition, oxidative stress is considered as an important factor in the etiology of AD and a multitude of metalloproteins and transporters is affected, leading to metal ion misregulation. Redox‐active metal ions (e.g., copper) can catalyze the production of reactive oxygen species (ROS) in the presence of molecular oxygen and a reductant such as ascorbate. The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative stress conditions. Thus, detecting ROS in vivo or in biological models of AD is of interest for better understanding AD etiology. The use of biocompatible and highly specific and sensitive probes is needed for such a purpose, since ROS are transient species whose steady‐state concentrations are very low. Luminescent lanthanide complexes are sensitive probes that can meet these criteria. The present review focuses on the recent advances in the use of luminescent lanthanide complexes for ROS biosensing. It shows why the use of luminescent lanthanide complexes is of particular interest for selectively detecting ROS (O2·‐, HO•, 1O2, H2O2, etc.) in biological samples in the µM‐nM range. It particularly focuses on the most recent strategies and discusses what could be expected with the use of luminescent lanthanide complexes for better understanding some of the molecular mechanisms underlying the development of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmacological treatments targeting calcitonin gene‐related peptide (CGRP) signalling molecules have provided safe and effective treatments, monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit beneficial, it is important to understand the molecular mechanisms of these new drugs to better understand migraine pathophysiology and improve therapy. Here, we describe current views on the role of the CGRP family of peptides ‐ CGRP, calcitonin, adrenomedullin, amylin ‐ and their receptors in the trigeminovascular system. All these molecules are present within the trigeminovascular system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc [ABSTRACT FROM AUTHOR]

Antibodies elicited by a synthetic immunogen, (Glu60Ala40)n -[Ala-DGlu(Lys-DAla-DAla)]5.1, related to a major class of peptidoglycan precursors, were purified by an affinity column of Sepharose 4B, to which the hapten, Ala-DGlu(Lys-DAla-DAla), was covalently attached and were then coupled to Sepharose 4B. Low-molecular-weight [14C]alanine-labeled products secreted by Micrococcus luteus cells grown in the presence of penicillin G were applied to the antibody-coupled gel. The bound material, which should be univalent to the antibody, was eluted completely by 0.02 mM α-tert-butyl- oxycarbonyl-lysyl-D-alanyl-D-alanine. High-molecular-weight uncross-linked soluble peptidoglycan secreted by M. luteus cells grown in the presence of penicillin G was completely bound to the affinity column, regardless of whether the soluble peptidoglycan was labeled in its alanine moieties or in its glucosamine and muramic acid residues A minor fraction of the bound soluble peptidoglycan was released with 0.02 mM tripeptide (‘paucivalent’ fraction). The remainder of the bound material was eluted with 2 mM tripeptide, indicating that a major portion of the high-molecular-weight material was ‘multivalent’. When glycan-labeled soluble peptidoglycan was stored in buffered saline at -5 °C for several months, a fraction of the labeled material was no longer bound to the affinity gel. Digestion of this non-binding fraction, as well as the paucivalent and multivalent fractions with hen egg-white lyso- zyme, was consistent with the affinity chromatographic data. These results indicate that an amidase present in these preparations cleaved different amounts of peptide from the soluble peptidoglycan preparations, producing strands which are polydisperse with respect to their peptide substitution. [ABSTRACT FROM AUTHOR]

The primary structure of the most acidic (pI = 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N-bromosuccinimide cleavage of the protein. The amino acid sequence so obtained is considered in comparison with those know for other parvalbumins and for rabbit troponin-C. [ABSTRACT FROM AUTHOR]

Although the formation mechanism of biogenically metallic nanoparticles is broadly associated to enzyme mediation, major attention has been given to the role of proteins and peptides in oxido‐reduction of metallic ions leading to these nanostructures. Among the wide range of biomolecules that can act not only as capping agents but also as non‐enzymatic agents to form nanoparticles, disulphide bridge‐containing peptides and amino acids particularly stand out. The literature proposes that they actively participate in the process of nanoparticles' synthesis, with thiols groups and disulphide bridge moieties as the reaction catalytic sites. Similarly, denaturated enzymes containing exposed S–S or S–H moieties are also able to reduce metallic ions to form nanoparticles. This mini‐review is focused on the biogenic synthesis of metallic nanoparticles such as gold, silver, copper, platinum, palladium, lead and selenium, in which proteins, peptides, reductases and even oxido‐reductases act as non‐enzymatic catalysts of the reduction reaction, opening economically and ecologically favourable perspectives in the nanoparticles synthesis field. [ABSTRACT FROM AUTHOR]

The recent discovery of new potent therapeutic molecules that do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). CPPs were first discovered based on the potency of several proteins to enter cells. Numerous CPPs have been described so far, which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization and the second involving formation of stable, non-covalent complexes with drugs. Nowadays, CPPs constitute very promising tools for non-invasive cellular import of cargo and have been successfully applied for in vitro and in vivo delivery of therapeutic molecules varying from small chemical molecule, nucleic acids, proteins, peptides, liposomes and particles. This review will focus on the structure/function and cellular uptake mechanism of CPPs in the general context of drug delivery. We will also highlight the application of peptide carriers for the delivery of therapeutic molecules and provide an update of their clinical evaluation. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (αβγδ)4. An intrinsic calmodulin (CaM, theδ subunit) binds directly to theγ protein kinase chain. The interaction site of CaM onγ has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed.15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical‘compact’ CaM complex. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiaeis a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease inS. cerevisiae.It is encoded by theCYM1gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption ofCYM1not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71–77)catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp,Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. TheL. vannameihistone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition againstMicrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µmwhile a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. [ABSTRACT FROM AUTHOR]

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of ‘sweet fingers’, i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential ‘sweet fingers’ of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37–47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the ‘wedge model’, recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett. 526, 1–3.]. [ABSTRACT FROM AUTHOR]

The preprotein transtocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein transtocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as welt as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of n-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein transtocation pore at the inner envelope membrane. [ABSTRACT FROM AUTHOR]

A Sin3-interacting domain (SID) originally described in Mad proteins is necessary for both transcriptional repression and growth suppression by these transcription factors. We recently reported that a structurally and functionally related Mad1-like SID is also present in five Sp1-like repressor proteins (TIEG1, TIEG2, BTEB1, BTEB3 and BTEB4), demonstrating that SID-mSin3A interactions have a wider functional impact on transcriptional repression. SID-mSin3A interaction is necessary for the anti-proliferative function of Mad, TIEG and BTEB proteins. It remains to be established, however, whether the SID-mSin3A interaction is constitutive or regulated. Here, we describe that the Mad1-like SID domain of the Sp1-like repressor TIEG2 is inhibited by the epidermal growth factor (EGF)-Ras-MEK1-ERK2 signaling pathway, via phosphorylation of four serine/threonine sites adjacent to the SID. This phenomenon disrupts the SID-mSin3A interaction and thereby inhibits TIEG2's repression activity. Thus, these results show for the first time that the repression of a SID-containing protein is regulated by signaling rather than functioning in a constitutive manner, extending our understanding of how the function of SID-containing repressors may be controlled. [ABSTRACT FROM AUTHOR]

Fungal conventional kinesins are unusually fast microtubule motor proteins. To compare the functional organization of fungal and animal conventional kinesins, a set of C-terminal deletion mutants of the Neurospora crassa conventional kinesin, NcKin, was investigated for its biochemical and biophysical properties. While the shortest, monomeric construct comprising the catalytic core and the neck-linker (NcKin343) displays very high steady-state ATPase (kcat = 260/s), constructs including both the full neck and adjacent hinge domains (NcKin400, NcKin433 and NcKin480) show wild-type behaviour: they are dimeric, show fast gliding and slower ATP turnover rates (kcat = 60-84/s), and are chemically processive. Unexpectedly, a construct (NcKin378, corresponding to Drosophila KHC381) that includes just the entire coiled-coil neck is a monomer. Its ATPase activity is slow (kcat = 27/s), and chemical processivity is abolished. Together with a structural analysis of synthetic neck peptides, our data demonstrate that the NcKin neck domain behaves differently from that of animal conventional kinesins and may be tuned to drive fast, processive motility. [ABSTRACT FROM AUTHOR]

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGα activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGγ only activates cleavage after basic residues. We have isolated REGγ mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome β subunits. The most robust REGγ specificity mutants involve substitution of Glu or Asp for Lys188. REGγ(K188ELD) variants are virtually identical to REGα in proteasome activation but assemble into less stable hepta- mers/hexamers. Based on the REGα crystal structure, Lys188 of REGγ faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin- like subunits by 125I-YL3-VS demonstrates that REGγ(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGγ(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGγ molecule. [ABSTRACT FROM AUTHOR]

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coti RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shill assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C-terminus or from N-terminal residues 6–39. A peptide corresponding to amino acid positions 152–169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303–353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA-binding domain of the RecA protein. [ABSTRACT FROM AUTHOR]

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the α- and β-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of α-subunit linked to a 54-amino-acid endopeptide and β-subunit was lower than with the subunits alone. [ABSTRACT FROM AUTHOR]

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin α and thymosins β4 and β9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin α, rat parathymosin and calf thymosin β4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin α and rat parathymosin both migrate to the nucleus whilst thymosin β4 remains in the cytoplasm. The peptide (1 - 88) of calf prothymosin α is shown not to accumulate in the Xenopus nucleus, demonstrating that the C- terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin β4 should be grouped separately from the pro- and parathymosins. [ABSTRACT FROM AUTHOR]

The gene encoding fimA, a 36 kDa fimbrial adhesin of Streptococcus parasanguis FW213, is highly conserved in all four genetic groups of sanguis streptococci. FimA-like peptides were produced by all strains tested. The nucleotide sequence directly upstream of fimA contains two open reading frames, ORF5 and ORF1, whose deduced protein products are homologous to members of a superfamily of ATP-binding cassette membrane transport proteins, including both prokaryotic and eukaryotic uptake and export systems. The amino acid sequence of FimA contains the consensus prolipoprotein cleavage site (LxxC) common to the 'periplasmic' binding proteins of Gram-positive transport systems. The deduced product of ORF5 is a 28.6 kDa membrane-associated protein that has the consensus binding site for ATP (GxxGxGKS). It shares significant homology with AmiE of Streptococcus pneumoniae as well as with Escherichia coli proteins involved in iron(lll) uptake. Allelic-replacement mutagenesis of ORF5 resulted in greatly increased resistance to aminopterin. These data demonstrate functionality with the amiE locus as well. The deduced product of ORF1 is an extremely hydrophobic integral membrane protein of 30.8 kDa with a pattern of six potential membrane-spanning regions, typical of a component of these types of transport system. The nucleotide sequence downstream of fimA, ORF3, encodes a 20 kDa protein having 78% identity with the 20 kDa protein encoded downstream of ssaB, a fimA homologue in S. sanguis 12. It also exhibits significant homology with bacterioferritin co-migratory protein (Bcp) of E. coli K-12. Allelic-repiacement mutagenesis in the fimA locus of FW213 showed that (i) expression of fimA was initiated at a site far upstream of the fimA start codon, and (ii) expression of fimA was not linked to expression of ORF3. Northern blots probed with... [ABSTRACT FROM AUTHOR]

The carbohydrate structure and complete amino acid sequence of a human lambda-type immunoglobulin light chain, protein Sm Z, has been determined. The protein was isolated from the urine of a patient with a plasma cell dyscrasia resembling γ-heavy-chain disease. 13 tryptic peptides covering the entire polypeptide chain of 135 residues were isolated from the aminoethylated protein, and 15 chymotryptic peptides, accounting for 131 residues, were recovered from the carboxymethylated protein. The sequence of 18 of these peptides was partially or completely determined by the Edman-dansyl technique or C-terminal analysis, permitting the establishment of the complete primary structure of the polypeptide chain. The sequence established that this light chain possessed an intramolecular deletion of 81 amino acid residues. The N-terminal 30 residues showed considerable homology with other lambda chains of subgroup II. The defect began at position 31, in the first hyper-variable region, and encompassed the remainder of the variable region through position 109. The constant region was fully intact and normal synthesis recommenced with a glutaminyl residue at position 110, the first residue of the constant region. This light chain contained carbohydrate in the hyperyanable region just preceding the deletion. The precise number and locations of the oligosaccharide chains were established by amino acid sequence analysis of glycopeptides isolated from proteolytic hydrolysates by chromatography on Bio-GeI P-6 columns. These studies showed that protein Sm A contains one N-glycosidically-linked chain attached to asparagine-25 and one O-glycosidically-linked oligosaccharide chain attached to serine-21. The structures of the oligo-saccharide chains were determined by methylation analysis, gas chromatography and hydrolysis with specific glycosidases. The structure of the N-glycosidically-linked chain was NeuAc(α2-→6)Gal(β1→4)GlcNAc(β1→2)- Man(α1→6)[NeuAc(x2→6)Gal(β→4)GlcNAc(β1→2)Man(α1→3)]Man(β1→4)GlcNAc(β1→4)[Fucα1→6]-GlcNAc→Asn. The second O-glycosidicalIy-linked chain was a disialylated tetrasaccharide with the structure, Neu(x2→3)Gal(β1→3)[NeuAc(α2→6)GalNAC→Ser. This mucin-type disialylated tetrasaccharide in close proximity to N-asparagine-linked chains has not been previously observed in the oligosaccharide chains of immunoglobulins. [ABSTRACT FROM AUTHOR]