Enzymatic activity of the second component of complement.

Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-alpha-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex G-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C1s. The action was enzymatically mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X 10(-2) mol. Cleavage of C2 by C1s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic acitivity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C42 complex, since AcGlyLysOMe competitively inhibited the C42 mediated cleavage of C3 in free solution and the C42 dependent binding of C3 to cells.     

Isolation of the smallest component of silk protein.

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.     

Conversion of the alpha component of penicillin-binding protein 1b to the beta component in Escherichia coli.

Among components alpha, beta, and gamma of penicillin-binding protein 1b, the alpha and gamma components were confirmed to represent the primary gene products by agreement of their N-terminal amino acid sequences with those predicted from the nucleotide sequence of the ponB (penicillin-binding protein 1b) gene with exclusion of the first methionine in each component. The generation of beta occurred primarily after cell disruption, and the simultaneous loss of alpha suggested the conversion of alpha to beta. The N-terminal amino acid sequence analyzed for beta showed that the conversion was due to the removal of 24 amino acids from the N terminus of alpha.     

The recognition component of the N-end rule pathway.

The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues.     

Laccase component of the Ceriporiopsis subvermispora lignin-degrading system.

Laccase activity in the lignin-degrading fungus Ceriporiopsis subvermispora was associated with several proteins in the broth of cultures grown in a defined medium. Activity was not increased significantly by adding 2,5-xylidine or supplemental copper to the medium. Higher activity, associated with two major isoenzymes, developed in cultures grown on a wheat bran medium. These two isoenzymes were purified to homogeneity. L1 and L2 had isoelectric points of 3.4 and 4.8, molecular masses of 71 and 68 kDa, and approximate carbohydrate contents of 15 and 10%, respectively. Data indicated 4 copper atoms per mol. L1 and L2 had overlapping pH optima in the range of 3 to 5, depending on the substrate, and exhibited half-lives of 120 and 50 min at 60 degrees C. They were strongly inhibited by sodium azide and thioglycolic acid but not by hydroxylamine or EDTA. The isoenzymes oxidized 1,2,4,5-tetramethoxybenzene but not other methoxybenzene congeners. A variety of usual laccase substrates, including lignin-related phenols and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)], were also oxidized. Kinetic parameters were similar to those of the laccases of Coriolus versicolor. The N-terminal amino acid sequence (20 residues for L1) showed significant homology to those of laccases of other white rot basidiomycetes but not to those of the laccases of Agaricus bisporus or Neurospora crassa.     

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg(2+)-dependent and is stimulated by Mn(2+) and F(-) ions. The initial reaction is rapid, with a broad pH profile and a ;transition' temperature for its activation energy at 27 degrees C. The apparent K(m) for ATP is 5mum. ATP, N-ethylmaleimide, Cu(2+) ions and NaIO(4) are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2-3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.     

Group-specific component in Macaca.

The group-specific component in 17 Macaca mulatta was examined. All the individuals revealed the same Gc 1--1 pattern.     

Centrin is a component of the pericentriolar lattice.

Here, we use three polyclonal anticentrin antisera designated 08/28, 26/14-1, and 26/14-2 to further characterize the pericentriolar lattice of metazoan cells. All of these antibodies give an indistinguishable localization pattern that consists of a constellation of pericentrosomal spots. In QT6 cells these spots are few in number and closely associated with the centriolar region, whereas in PtK2 cells they are more numerous and distributed further from the point of microtubule focus. In mitotic cells, centrin is localized to the spindle poles and spindle apparatus. We demonstrate here that the pericentriolar lattice of PtK2 and QT6 cells is, in part, composed of proteins characterized by acidic pIs (4.4 to 5.4), low molecular mass (M(r) 18,500-21,000), and calcium-binding; these attributes and the immunoreactivity of these proteins to anticentrin antibodies indicate that they are centrin isoforms of metazoan cells. Finally, we confirm our earlier observation that PtK2 cells contain a centrin-related protein of M(r) 165,000; QT6 cells also contain centrin-related proteins (M(r) 64,000-165,000). We conclude that centrin is a component of the pericentriolar lattice of higher eukaryotic centrosomes.     

Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.     

The predicted structure of the calcium-binding component of troponin.

The structure prediction of the calcium binding component of troponin (TN-C) incorporates the following assumptions: (1) TN-C contains four regions homologous to the calcium binding "EF hand" of parvalbumin. (2) The four EF hands are arranged in two pairs with overall symmetry, 222. (3) The regions of the calcium binding component of troponin which are not in the four EF hands connect the hands within each pair, one to two and three to four, and connect the pairs, region two to region three. In the resulting model there is a well-defined hydrophobic core made from side chains of all eight helical regions and of the four calcium binding loops. The Ca2+ within pairs are separated by 11 A; while the pairs of Ca2+ are separated from one another by over 30 A. Cys-98 and Tyr-109 are suggested to be sensitive spectroscopic probes. Calcium(1) is suggested to be solvent accessible and most readily replaced by a lanthanide. Because of the overall symmetry of the calcium binding component of troponin, one can anticipate that the inhibitory- and the tropomyosin binding components of troponin are similar to one another.     

The proteins of the keratin component of bird's beaks.

Birds' beaks have an outer shell of hard keratin which consists almost entirely of proteins which are very rich in glycine [about 30 residues per 100 residues (residues %)], contain moderate levels of tyrosine and serine (each about 8 residues %), and which have relatively low contents of cystine (about 2-5 residues %), lysine, histidine, isoleucine and methionine. Major protein fractions in the S-carboxymethyl form isolated from the beaks of six different orders of birds have similar amino acid compositions, isoelectric points (pH 4-2-4-9) and molecular weights (13,000-14,500). Detailed chromatographic electrophoretic and compositional studies of the proteins of kookaburra beak reveal them to be a family of closely related proteins with only limited heterogeneity, in contrast to mammalian keratin systems. The major kookaburra beak fraction is similar in overall composition and molecular weight to fowl epidermal scale, kookaburra claw and turtle scute proteins and shows some resemblance to reptile claw protein. Beaks also contain small amounts of protein which are distinctly different from the major fraction but which resemble feather keratin proteins in composition and size.     

MotX, the channel component of the sodium-type flagellar motor.

Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.     

Gamma-tubulin is a highly conserved component of the centrosome.

We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.     

A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.