Theo Hofmann

A monoclonal immunoglobulin G1 in which some molecules possess glycosylated light chains--I. Site of glycosylation.

Abstract A human monoclonal IgG1 κ protein (Hom) was found to possess two species of light chain with molecular weights of 23,000 and 28,000 respectively. The difference in mass was due to the presence of a carbohydrate prosthetic group on the 28,000 dalton species. The two species of light chain were separated by chromatography on CM-cellulose. Circular dichroism failed to detect any significant differences in conformation between the glycosylated and non-glycosylated forms and both species recombined with a heavy chain in an identical fashion as judged by difference spectroscopy. An anti-idiotypic antibody raised against the glycosylated Fab-fragment of IgGlHom was unable to distinguish between this species and the non-glycosylated Fab fragment, suggesting that the structure of the combining site is identical or very similar in both molecules. Amino-acid sequence analyses showed that both light chains had an identical sequence up to residue 41. Further studies on the glycosylated light chain, using o -iodosobenzoic acid fragments, localized the carbohydrate attachment site to asparagine 107, the penultimate residue of the J κ region which links V κ to C κ . The sequence around the attachment site was Asn-Arg-Thr which satisfies the requirements for an acceptor sequence. The non-glycosylated light chain was found to have the same sequence in this region indicating that the absence of glycosylation was not due to the lack of an acceptor sequence. A kinetic mechanism has been proposed to account for the incomplete glycosylation of the light chains of IgG1Hom in which there is a competition between the rate of folding of the nascent polypeptide and the rate of attachment of core sugars via the dolichol pyrophosphate pathway.

Acyl intermediates in pepsin and penicillopepsin catalyzed reactions.

Summary Porcine pepsin and penicillopepsin catalyze transpeptidation reactions which involve the transfer in high yield of N-terminal leucine from Leu-Tyr-Leu, and Leu-Tyr amide to intact substrate with the subsequent release of Leu-Leu. With the latter substrate porcine pepsin also forms Leu-Leu-Leu. With penicillopepsin small amounts of Leu-Leu-Tyr-Leu and Leu-Leu-Tyr amide resp. were obtained as intermediates from Leu-Tyr-Leu and Leu-Tyr amide. No radioactivity was incorporated into Leu-Leu when Leu-Tyr-Leu and [14C]-leucine (1:6) were incubated with the enzymes. It was concluded that the transpeptidation proceeds via covalent acyl intermediate.

Activation of the action of penicillopepsin on leucyl-tyrosyl-amide by a non-substrate peptide and evidence for a conformational change associated with a secondary binding site.

Abstract The cleavage of L-leucyl-L-tyrosyl amide by penicillopepsin is activated about tenfold by L-leucyl-glycyl-L-leucine. The latter is not a substrate. The activator has no effect on K M . An activation constant K A = 2.0 ± 0.6 mM has been calculated. Leucyl-glycyl-leucine also affects four bands of the circular dichroism spectrum of the enzyme. A dissociation constant of 2.4 mM has been calculated from a titration of the ellipticity changes. The results suggest that a conformational change caused by binding of the peptide is responsible for the increased catalytic activity.

At-sea observations of ivory gulls ( Pagophila eburnea ) in the eastern Canadian high Arctic in 1993 and 2002 indicate a population decline

Evidence from colony surveys and local Inuit knowledge strongly suggest that the Canadian population of ivory gulls ( Pagophila eburnea ) has declined dramatically. The observations of ivory gulls at sea presented here are consistent with this. Ivory gulls were observed during two cruises on the Russian icebreaker Kapitan Khlebnikov in the eastern Canadian high Arctic in August 1993 and 2002. Ivory gulls were seen 3.5 times more often in 1993 (n = 176) than in 2002 (n = 149), and, corrected for observation effort, four times more ivory gulls were seen in 1993 than in 2002. Ivory gulls are scavengers: they were never observed feeding on fish behind the vessel while ice-breaking, although black-legged kittiwakes ( Rissa tridactyla ) often were seen feeding in this way. Ivory gulls were observed scavenging around polar-bear ( Ursus maritimus ) kills in 1993 but not in 2002. By far the largest number of ivory gulls was seen near Grise Fiord in 1993. There, opportunities for them to scavenge were likely good at the community landfill as well as at Inuit and polar-bear kills due to complete ice coverage of the surrounding marine area. No ivory gulls were seen there in 2002. Observations of four individuals in 1993 and five individuals in 2002 near the southern end of Eureka Sound and in Norwegian Bay, 150 km from the nearest known breeding colonies, suggest that as yet undiscovered colonies might exist in this area. With three lines of evidence (colony surveys, local Inuit knowledge, at-sea surveys) now indicating population decline, urgent reassessment of the status of ivory gulls in Canada needs to take place.

Penicillopepsin: 2.8 a Structure, Active Site Conformation and Mechanistic Implications

The crystal structure of penicillopepsin, an extracellular acid protease isolated from the mold Penicillium janthinellum, has been determined at 2.8 A resolution by the method of multiple isomorphous replacement. The resulting electron density map computed from the native structure factor amplitudes and MIR phases has an overall mean figure of merit of 0.90. The molecule is decidedly nonspherical, with the majority of residues in beta-structure. There is an 18-stranded mixed beta-sheet which forms the structural core in the region of the active site. This site, identified by the covalent binding of two EPNP molecules to Asp-32 and Asp-215, is located in a deep groove which divides the molecule into two approximately equal lobes. Both aspartic acid residues in the active site are in intimate contact with one another and the carboxyl group of Asp-32 makes two other important hydrogen-bonded contacts: one with Ser-35 and the other with the main chain peptide bond between Thr-216 and Gly-217. A proposed mechanism for acid protease catalysis is similar in many aspects to that proposed for carboxypeptidase A. The electrophilic component which polarizes the substrate carbonyl bond in the acid proteases is the proton shared between the beta-carboxyl groups of Asp-32 and Asp-215. The beta-carboxyl group of Asp-32 removes a proton from a water molecule bound between this side chain and the substrate; the resultant OH- attacks the carbonyl carbon atom of the substrate molecule. The phenolic -OH group of Tyr-75 donates its proton to the amide nitrogen of the scissile bond of the substrate.

The primary structure of carboxypeptidase S1 from Penicillium janthinellum

The complete amino acid sequence of carboxypeptidase Sl from Penicillium janthinellum has been determined by N‐terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleavage with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu‐specific proteinase from B. licheniformis. The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half‐cystine residues and one glycosylated asparagine residue. A comparison to other carboxypeptidases shows that the enzyme is homologous to carboxypeptidase‐Y and carboxypeptidase‐MIII from malt. Specificity and binding of substrates are discussed from a three‐dimensional model based on the known structure of carboxypeptidase‐Y from Saccharomyces cereviciae and carboxypeptidase II from wheat.

Viral membrane glycoproteins: Comparison of the amino acid terminal amino acid sequences of the precursor and mature glycoproteins of three serotypes of vesicular stomatitis virus

The NH2-terminal amino acid sequences of the envelope glycoproteins and the in vitro synthesized, nonglycosylated precursors of the glycoproteins of three serotypes, namely Indiana (Toronto), Cocal, and New Jersey (Concan) of vesicular stomatitis virus were determined. A comparison of the sequences showed little homology in the signal peptides present in the nonglycosylated precursors except for their high hydrophobic amino acid content. In contrast, the NH2-terminal amino acid sequences of the mature envelope glycoproteins revealed extensive homology suggesting that this region is conserved and may be involved in essential biological function(s) of the rhabdovirus.

The use of penicillocarboxypeptidase-S1 in amino acid sequencing

Abstract The action of penicillocarboxypeptidase-S1 on a number of peptides obtained from enzymatic digests of penicillopepsin and other sources has been studied and compared with that of pancreatic carboxypeptidase A. In addition to proline, lysine, and arginine, penicillocarboxypeptidase-S1 readily cleaved glycine, and glutamic and aspartic acids from peptides which were hydrolyzed only slowly or not at all by carboxypeptidase A. Penicillocarboxypeptidase-S1 was also useful in locating the charges in peptides which contained several residues of aspartic and glutamic acids and their amides. The enzyme did not cleave γ- l -glutamyl bonds in synthetic peptides, but liberated γ- l -glutamyl- l -alanine from γ- l -glutamyl-γ- l -glutamyl- l -alanyl-γ- l -glutamyl-alanine. Although there were considerable differences in the rate at which different peptides could be degraded, this rate is not determined by the C-terminal amino acid alone. Generalizations cannot be made at this stage but it appears that some dipeptide sequences made up of two of the following—glycine, alanine, serine, threonine, and proline—were cleaved considerably more slowly than other bonds. Dipeptides with a free N-terminal were hydrolyzed very slowly, if at all. Peptides containing d -amino acids were not hydrolyzed. Penicillocarboxypeptidase-Sl appears to be a very useful enzyme in sequence work.

Identification of the iodine-sensitive tyrosines in porcine pepsin☆

Porcine pepsin was iodinated at pH 6.0 and 37° with a 13-fold molar excess of [125I]triiodide. Loss of proteolytic activity levelled off at 75 ± 5%, and 2.8 ± 0.1 gram atoms of iodine were incorporated per mole of enzyme. Pepsin, iodinated in this manner, was digested with chymotrypsin. The bulk of the label was located in two peptides with the sequences: Leu-Gly-Gly-Ile-Asp-Ser-Ser-diiodoTyr-Tyr and Ile-Gly-Asp-Glu-Pro-Leu-Asn-iodoTyr. The latter peptide is the N-terminal sequence of pepsin. It is postulated that one or both of these tyrosines may form part of the secondary binding site of pepsin.

A method for eliminating Rayleigh scattering from fluorescence spectra

A numerical method is described for the elimination of Rayleigh scattering from protein fluorescence emission spectra. The method is based upon the observation that Rayleigh scattering is symmetrical about a wavelength at or near the wavelength of excitation. It works best when an automated, computer-based approach can be applied to spectra collected on a data-logging device, although manual correction of chart-recorded spectra is also possible.