The sequence of equine muscle carbonic anhydrase

The sequence of equine muscle carbonic anhydrase (CA-III) has been determined. The 2 reactive cysteines of the 5 such residues have been localized. A strong sequence homology to other mammalian carbonic anhydrases exists, and 91% of the residues in the equine and bovine muscle forms are identical.     

Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein.

A dimeric glycoprotein, glucose oxidase, was allowed to react with lysine-specific cross-linkers, both when immobilized on a succinoylated lectin matrix at a critically low density and also at a high density in solution. Analysis of the cross-linked complexes thus obtained led to the following inferences with regard to the structure of this protein. (1) Of the 15 lysine residues on each glucose oxidase protomer, none is available on the non-interfacial surfaces. (2) Assuming that this protein possesses C2 symmetry with isologous bonding between subunits, it may be inferred that on each promoter there are at least two lysine clusters along or close to the interprotomeric interface. (3) These "interfacial' lysine residues on each protomer are so oriented that the epsilon-amino groups of lysine residues a and b on protomer 1 "face', and are very close to, the epsilon-amino groups of lysine residues b' and a' respectively on protomer 2. General inferences on the geometry of dimeric proteins derivable from an analysis of the cross-linked complexes obtained (as well as those not seen) by using this low-density matrix cross-linking approach were enumerated. Modified lectin matrices may prove useful in studying the three-dimensional structure of glycoproteins, particularly non-crystallizable oligomers.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Nonenzymatic incorporation of glucose and galactose into brain cytoskeletal proteins in vitro.

Initial studies demonstrated the loss of lysine and simultaneous appearance of glucitollysine in intracellular proteins following incubation with sugar. For example, when a crude nervous tissue cytoskeletal preparation was incubated in 100 mM glucose for 10 days, > 60% of the lysine residues were modified. Over 20% of the lysyl residues in a spinal cord neurofilament preparation are susceptible to Schiff base formation after one day and over 30% following five days of incubation with 100 mM glucose. When incubated with 100 mM galactose, F- and G-actin were found to be significantly modified in as few as 15 h, with > 70% of the lysyl residues lost. After 45 h of incubation, > 90% of the residues had been modified. These data also indicate that many of the lysyl residues in F- and G-actin are exposed and very susceptible to modification by sugar. This rapid and extensive modification of lysine in actin in vitro suggest that it may be modified in diabetic nervous tissue.     

A spectrophotometric assay for the characterization of the S'' subsite specificity of alpha-chymotrypsin

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.     

Comparative immunochemical studies of carbonic anhydrase III in horses and other mammalian species

1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.     

Isolation of equine muscle carbonic anhydrase in crystalline form

A relatively simple procedure for the isolation of equine muscle carbonic anhydrase (CA-III) in crystalline form has been developed. An important aspect of the method, which should find ready application to this enzyme from other species, is the alkylation of the readily reactive cysteine residues prior to starting the fractionation.     

Substrate preference of transglutaminase 2 revealed by logistic regression analysis and intrinsic disorder examination

Tissue transglutaminase (TG2) catalyzes the Ca²⁺-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database†, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.     

WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain

WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues serving as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Functional similarities with rabbit muscle aldolase.

Fructose diphosphate aldolase of Mycobacterium smegmatis is found to be a class I type aldolase and possesses functional similarities with rabbit muscle aldolase with respect to the amino acid residues at the catalytic site. The presence of a lysine residue at the active site is indicated by the formation of a Schiff-base with the substrate. The lower degree of inactivation compared to rabbit muscle aldolase on treatment with carboxypeptidase-A suggests the absence of an essential terminal tyrosine residue. Participation of histidine residues in enzyme catalysis is suggested by the photoinactivation of the enzyme in presence of methylene blue. Finally, thiol groups do not seem to have a direct role in catalysis.     

Chemistry of the collagen cross-links. Nature of the cross-links in the polymorphic forms of dermal collagen during development.

Both the type I and type III collagens present in embryonic dermis are stabilized by the intermolecular cross-link, hydroxylysino-5-oxonorleucine, derived from hydroxylysine-aldehyde, although the type I collagen possesses a significant proportion of dehydrohydroxylysinonorleucine. However, concurrent with the change in the proportion of the two types of collagen during postnatal development there is a change-over with both type I and III collagens to the labile cross-link, dehydrohydroxylysinonorleucine, derived from lysine aldehyde. The results indicate that the change in the nature of the cross-link with development is determined primarily by the change in the extent of hydroxylation of the lysine residues in the terminal non-helical regions rather than being due to the change in the type of collagen.     

Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design

We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme–inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl–substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.     

Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level

The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.

Involucrin is a 68-kDa precursor of the keratinocyte cornified envelope. During keratinocyte terminal differentiation glutamine residues of involucrin become covalently cross-linked to other envelope precursors via covalent epsilon-(gamma-glutamyl)lysine bonds. In the present study we examine the secondary and tertiary structure of human involucrin using computer algorithms, circular dichroism, and electron microscopy. Our results indicate that involucrin is an extended, flexible, rod-shaped molecule that has a length of 460 A, an axial ratio of 30:1 and possesses between 50 and 75% alpha-helical content. Glutamine residues are circumferentially distributed along the length of the alpha-helical segments of the molecule, a distribution that is conserved in all species. We hypothesize that this distribution of glutamine residues together with the elongated shape of the molecule permits optimal interaction of involucrin glutamyl side chains with the lysine residues of other para-membranous proteins during transglutaminase-mediated cross-linking. Moreover, its long length allows involucrin to cross-link molecules that are separated by substantial distances in the cornified envelope. These properties allow a single involucrin molecule to form multiple cross-links, in multiple spatial planes, with other envelope precursors. Thus, the structure of involucrin is that of an ideal intermolecular cross-bridge.     

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.     

Evidence for the presence of carbonic anhydrase III in the myoid cells of the bovine thymus. Immunocytochemical and ultrastructural study

An immunohistochemical study was carried out to detect the localization of carbonic anhydrase III (CA-III) in the bovine thymus. It was found that the CA-III activity was localized in the cells forming small clusters dispersed in the medullary region. By ultrastructural observation, these cells were identified as myoid cells.     

Localization of lysines acetylated in ubiquitin reacted with p-nitrophenyl acetate

The protein ubiquitin undergoes extensive N epsilon-acetylation of some of its seven lysine residues when reacted with p-nitrophenyl acetate. Lysines 27 and 29 show little reactivity whereas residue 6 is the most readily acetylated. Residues 11, 33, 48, and 63 show intermediate reactivities.     

Comparative immunohistolocalization of carbonic anhydrase isozymes I, II and III in the equine and bovine digestive tract

Summary Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.