bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified branched‐chain amino acid pathways give rise to acyl acids of sucrose esters exuded from tobacco leaf trichomes
Authors: KANDRA Lili , SEVERSON Ray , WAGNER George Joseph
Published: March 2005

A major diversion of carbon from branched‐chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6‐O‐acetyl‐2,3,4‐tri‐O‐acyl‐α‐D‐glucopyranosyl‐β‐D‐fructofuranosides) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3‐methylvaleric acid, 2‐ and 3‐methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3–C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched‐chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3‐methylvaleryl and 2‐methylbutyryl groups are derived from the Thr pathway of branched‐chain amino acid metabolism, 3‐methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl‐CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids.

Results suggest that the shunting of carbon away from the biosynthesis of Val, Leu and Ile may be due to a low level of amino acid utilization in protein synthesis in specialized glandular head cells of trichomes. This would result in the availability of corresponding oxo acids for CoA activation and esterification to form sucrose esters. Preliminary evidence was found for the involvement of cycling reactions in oxo‐acid‐chain lengthening and for utilization of pyruvate‐derived 2‐oxobutyrate to form straight‐chain acyl substituents.
The role of carbohydrate in recombinant human erythropoietin
Authors: TSUDA Eisuke , KAWANISHI Gosei , UEDA Masatsugu , MASUDA Seiji , SASAKI Ryuzo
Published: March 2005

Recombinant human erythropoietin has N‐linked sugar [Tsuda et al., (1988) Biochemistry 27, 5646–5654]. Here we have demonstrated the presence of O‐linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Galβ(1–3)GalNAc.

To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoiein from which N‐linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N‐linked sugar abolished the in vivo biological activity completely, indicating that the presence of N‐linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin.

Incubation of asialo‐erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 °C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.
Preparation of monoclonal antibodies to hirudin and hirudin peptides
Authors: SCHLAEPPI Jean‐Marc , VEKEMANS Sylvie , RINK Hans , CHANG Jui‐Yoa
Published: March 2005

A panel of four monoclonal antibodies was obtained against hirudin, a potent and specific inhibitor of thrombin, by immunizing three groups of mice with protein conjugates made of recombinant desulfatohirudin (group I) or two synthetic peptides representing the C‐terminal sequences 40–65 (group II) and 52–65 (group III) of hirudin. Only the monoclonal antibody 4049‐83‐12, obtained from the group I of mice, showed high affinity for hirudin (Kd of 0.6 nM) and in vitro neutralizing properties. The anti‐peptide monoclonal antibodies bound hirudin with lower affinity (Kd of 1.5–7 nM) and showed lower neutralizing capacities. An epitope analysis performed by competitive ELISA using various hirudin analogues and by limited proteolysis of the hirudin‐antibody complex revealed that the binding domains of all the anti‐peptide antibodies were located close to the C‐terminus of hirudin, since the bond between Glu‐61 and Glu‐62 was not cleaved by the V8 staphylococcal protease in the presence of these antibodies. The epitope of the antibody 4049‐83‐12 was strictly conformation‐dependent, it recognized neither S‐carboxymethylated hirudin nor any peptides of hirudin. The cleavage of the bond between Glu‐43 and Gly‐44 by V8 protease, as well as the cleavage of the bond between Lys‐47 and Pro‐48 by lysyl endopeptidase, was prevented by the binding of the antibody 4049‐83‐12 to hirudin. The possibility that this epitope overlapped with a region of hirudin involved in the binding to thrombin is discussed.
The behavior of proteases in lecithin reverse micelles
Authors: PENG Qiaoqian , LUISI Pier Luigi
Published: March 2005

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and α‐chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co‐surfactant. The C8‐lecithin system, i.e. 1,2‐dioctanoyl‐sn‐glycero‐3‐phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to wo 60, where wo= [H2O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 1H‐NMR; the proton chemical shift as a function of wo was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2‐ethylhexyl sulfosuccinate). 31P‐NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and α‐chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (kcat and Km have been determined for a few systems). The bell‐shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell‐shaped curves are also obtained when studying the influence of wo on the enzyme activity, with an optimal wo which is in the range 7–10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.
Effects of ageing on transcriptional and post‐transcriptional regulation of malic enzyme and glucose‐6‐phosphate dehydrogenase in rat liver
Authors: FUKUDA Hitomi , KATSURADA Akihiko , IRITANI Nobuko
Published: March 2005

We previously found an age‐dependent impairment of induction of lipogenic enzymes in rat liver [Iritani et al. (1981) Biochim. Biophys. Acta 665, 636–639]. Further, we have found that after refeeding a fat‐free diet to fasted rats, increases in transcriptional rate, mRNA concentration and enzyme induction of hepatic malic enzyme and glucose‐6‐phosphate dehydrogenase were always lower in 18‐month‐old rats than in 1.5‐month‐old rats. In the young rats, the transcriptional rates reached the maximum level in 4 h and the mRNA reached maximum levels in 16 h. The peaks tended to delay in the older rats. The half‐lives of the mRNAs were not significantly longer in the old than in the young animals. The incorporation of [3H]leucine into the enzyme proteins was also decreased roughly in proportion to the enzyme induction. The mRNA concentrations in the liver polysomes were roughly proportional to the total mRNA. Thus, no effects of ageing on mRNA stability or on the translational activity of the enzymes could be found. It is suggested that the age‐dependent decreases of malic enzyme and glucose‐6‐phosphate dehydrogenase induction can be mainly ascribed to the transcriptional steps. Moreover, the transcriptional rate, mRNA concentration and induction of malic enzyme were increased by triiodothyronine treatment at a similar rate in both the young and old rats, but the absolute increments were lower in the old animals. The triiodothyronine response to malic enzyme induction also appeared to be primarily decreased at the transcription level.
Cloning and sequencing of mammalian glutathione reductase cDNA
Authors: TUTIC Mevlida , LU Xiang , SCHIRMER R. Heiner , WERNER Dieter
Published: March 2005

The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full‐length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated λGRM‐B11 was isolated by plaque‐screening of an induced mouse cDNA expression library in the λgt11 vector with a rabbit antibody probe to human glutathione reductase. 125Iodine‐labelled whole anti‐rabbit immunoglobulin was used as second antibody. EcoRI digestion of the λGRM‐B11 clone released a 720‐bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with λGRM‐B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti‐(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM‐B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM‐B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318.

The insert of the pGRM‐B11 plasmid was used as a bona fide nucleic acid probe to screen mouse and human cDNA libraries prepared in the λgt11 or in the λgt10 vector. The first full‐length cDNA clone (λGRH‐Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH‐B) is colinear with the mouse sequence cloned in the pGRM‐B11 plasmid. The fragment of intermediate size (pGRH‐CT) comprises the 3′ end of the mRNA and the poly(A) tail while the short fragment (pGRH‐NT) corresponds to the 5′ region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.
Purification and characterization of a cytosolic protein‐tyrosine kinase from porcine spleen
Authors: KOBAYASHI Tomoko , NAKAMURA Shun‐ichi , TANIGUCHI Takanobu , YAMAMURA Hirohei
Published: March 2005

A cytosolic protein‐tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S‐200, casein‐Sepharose 4B, heparin‐Sepharose CL‐6B and anti‐(4‐aminobenzyl phosphonic acid)– Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver‐stained band of molecular mass 40 kDa. The 40‐kDa cytosolic protein‐tyrosine kinase was purified approximately 10000‐fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometory of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 μM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40‐kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40‐kDa kinase described here is a different type of protein‐tyrosine kinase than the enzymes so far reported.
Effect of pressure and calcium on the reversible inhibition of the sarcoplasmic‐reticulum calcium‐transport enzyme and on its tryptic cleavage pattern
Authors: RONZANI Nelly , HASSELBACH Wilhelm , STEPHAN Lore
Published: March 2005

The reversible inhibition of the sarcoplasmic‐reticulum calcium‐transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium‐transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1 – 100.0 MPa affects trypsin cleavage of the control substrate N‐α‐benzoyl‐L‐arginine‐4‐nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium‐transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T1 cleavage site [C. J. Brandl et al. (1986) Cell 44, 597–607]. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1–3 in addition to the established peptides A1 and A2. Under pressure and in the presence of calcium, A1 and A2 rapidly disappear indicating the presence of calcium‐binding sites in these peptides. In contrast, the B1–3 peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as of the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium‐dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure‐induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20–40 ml/mol and 80–100 ml/mol in the ranges 0.1–40.0 MPa and 40.0–100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium‐transport enzyme which activates and modifies tryptic‐cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme resides in its E1 state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.
Localization of the peptidase activity of human serum butyrylcholinesterase in a – 50‐kDa fragment obtained by limited α‐chymotrypsin digestion
Authors: RAO Rammohan V. , BALASUBRAMANIAN Aiylam S.
Published: March 2005

Purified human serum butyrylcholinesterase (∼ 90‐kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited α‐chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of ∼ 50 kDa, ∼ 21 kDa and ∼ 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639–644] we characterized the ∼ 20‐kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the ∼ 50‐kDa fragment is characterized. This fragment, after isolation by Sephadex G‐75 chromatography from a chymotryptic digest of purified butyrylcholinesterase, exhibited only peptidase activity and was devoid of cholinesterase and aryl acylamidase activities. It could bind to a column of Ricinus communis agglutinin bound to Sepharose, indicating its glycosylated nature and the presence of galactose. The peptidase activity in the ∼ 50‐kDa fragment could be immunoprecipitated by a polyclonal antibody raised against purified butyrylcholinesterase. SDS‐gel electrophoresis of this fragment isolated by R. communis agglutinin – Sepharose and Sephadex G‐75 chromatography showed a protein band of ∼ 50 kDa by silver staining. Amino‐terminal sequence analysis of the ∼ 50‐kDa fragment gave the sequence of Gly‐Pro‐Thr‐Val‐Asp which corresponded to amino acid residues 291–295 in the butyrylcholinesterase sequence [Lockridge et al. (1987) J. Biol. Chem. 262, 549–557]. The combined results suggested that α‐chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a ∼ 20‐kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a ∼ 50‐kDa fragment exhibiting only peptidase activity.
A highly repetitive and transcribable sequence in the tortoise genome is probably a retroposon
Authors: ENDOH Hiroshi , NAGAHASHI Shigehisa , OKADA Norihiro
Published: March 2005

On in vitro transcription of total genomic DNA of the tortoise (Geoclemys reevessi), a discrete‐sized RNA of 6.5S was obtained that represented a highly repetitive and transcribable sequence in the tortoise genome. Three sequences of the 6.5S RNA gene were sequenced, and a consensus sequence was deduced from these three sequences and one reported previously [Endoh, H & Okada, N. (1986) Proc. Natl Acad. Sci. USA 83, 251–255]. The 5′ part of the gene showed close similaries to lysine (rabbit) and threonine (mouse) tRNAs (overall similarity 68–70%), so this tortoise sequence may have evolved from one of these tRNAs. The consensus sequence retained the expected CCA triplet at the 3′ end of tRNA, but not at the 3′ end of tDNA, supporting the idea that the tRNA‐related region of the gene was generated via an RNA intermediate. The 5′ and 3′ flanking sequences of the four genes were found to be completely different from each other. Fingerprint analysis and S1 nuclease mapping analysis also showed that sequence boundaries of tortoise repetitive units exactly corresponded to RNA species. These results, together with data obtained by Southern blot hybridization, indicated that the 6.5S RNA genes are dispersed in the tortoise genome. Therefore, generation of the tRNA‐related region of the gene and amplification of the whole unit of the gene are both RNA‐mediated events. The existence of this tortoise sequence suggests that short interspersed sequences are more common in eukaryotic genomes than had previously been thought.
Identification of three developmentally controlled isoforms of human myosin heavy chains
Authors: BOBER Eva , BUCHBERGER‐SEIDL Astrid , BRAUN Thomas , SINGH Surjit , GOEDDE Heinz Werner , ARNOLD Hans Henning
Published: March 2005

A set of cDNA clones coding for myosin heavy chains (MHC) was isolated from a human fetal skeletal muscle library. We have demonstrated by restriction mapping and nucleotide sequence analysis that the cDNAs represent three distinct transcripts, presumably the products of different genes. Furthermore, the pattern of mRNA expression indicates that the corresponding genes are regulated in a tissue‐specific and developmental‐stage‐specific manner. While the cDNA clone gtMHC‐V exhibits extensive sequence similarity to the rat β‐myosin heavy chain, the two other clones, gtMHC‐F and gtMHC‐E are very similar to the rat genes encoding the perinatal and embryonic myosin heavy chains, respectively. The mRNA corresponding to clone gt‐MHC‐V is highly expressed in heart and adult fast skeletal muscle and to a lesser extent in fetal skeletal muscle and adult slow skeletal muscle. The mRNAs corresponding to clones gtMHC‐F and gtMHC‐E are abundantly present in fetal skeletal muscle and are not present or barely detectable in heart and adult skeletal muscle.
Methylation of 23S ribosomal RNA due to carB, an antibiotic‐resistance determinant from the carbomycin producer, Streptomyces thermotolerans
Authors: ZALACAIN Magdalena , CUNDLIFFE Eric
Published: March 2005

A resistance gene, carB, originally isolated from the carbomycin‐producing organism, Streptomyces thermotolerans, confers on Streptomyces lividans high‐level resistance to the drug. However, ribosomes from S. lividans expressing car B show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The car B product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing erm E, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides.
Structure‐function relationships in the polypeptide cardiac stimulant, anthopleurin‐A
Authors: GOULD Alison R. , MABBUTT Bridget C. , NORTON Raymond S.
Published: March 2005

Selective proteolysis of the polypeptide cardiostimulant anthopleurin‐A by trypsin introduces a single break in the polypeptide backbone on the C‐terminal side of Arg14. The resulting derivative is devoid of any cardiostimulant activity. The structural changes which accompany this loss of activity have been examined by one‐ and two‐ dimensional 1H‐NMR spectroscopy. It is shown that the overall backbone folding of anthopleurin‐A is conserved on digestion, with some structural changes occurring for residues which are adjacent to the site of cleavage by trypsin. Thus, although previous NMR studies on anthopleurin‐A indicate that the region surrounding Arg14 is devoid of any ordered structure, it appears that some degree of structural integrity is required to allow the essential side chains to adopt the conformation necessary to produce a cardiostimulant effect.
Further studies on the role of glycogenin in glycogen biosynthesis
Authors: SMYTHE Carl , WATT Peter , COHEN Philip
Published: March 2005

About 90% of the glycogenin in skeleta muscle extracts prepared from fed, 24‐h starved or alloxan‐diabetic rabbits sedimented at 140 000xg with the glycogen/sarcovesicular fraction, from which it was released by glycogenolysis, but not by 1% SDS. Glycogenin in the glycogen/sarcovesicular fraction is therefore bound covalently to glycogen, and not associated (covalently or non‐covalently) with the sarcoplasmic reticulum. The same proportion of glycogen synthase was also recovered in the glycogen/sarcovesicle fraction, was solubilised by glycogenolysis, and copurified with glycogenin to yield a heterodimer composed of a 1:1 complex between these proteins. Glycogen synthase and glycogenin are therefore present in equimolar amounts in skeletal muscle and there is an average of one glycogen synthase catalytic subunit associated with each molecule of glycogen in vivo.

Glycogenin and glycogen synthase released into the muscle cytosol by degradation of glycogen did not form a complex initially, and only 50% reassociation took place after storage for several hours or overnight dialysis. This suggests that the muscle cytosol may contain a factor(s) which regulates glycogen biogenesis by modulating the association of glycogenin and glycogen synthase. Only glycogen synthase that was complexed to glycogenin was capable of elongating the primer formed by incubation of glycogenin with Mn2+and micromolar concentration of UDP‐glucose, demonstrating the critical importance of this complex for glycogen biogenesis.
Synthetic peptides as model substrates for the study of the specificity of the polycation‐stimulated protein phosphatases
Authors: AGOSTINIS Patrizia , GORIS Jozef , PINNA Lorenzo A. , MARCHIORI Fernando , PERICH John W. , MEYER Helmut E. , MERLEVEDE Wilfried
Published: March 2005

The substrate specificity of the different forms of the polycation‐stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg‐dependent (type 1) phosphatase (AMDc) was investigated, using synthetic peptides phosphorylated by either cyclic‐AMP‐dependent protein kinase or by casein kinase‐2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 Phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N‐terminal (Arg) basic residues. In contrast with the latter, the AMDc phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N‐terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDc phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)‐containing peptides may require specific determinants in the primary structure of the phosphorylation site.
Casein kinase II is elevated in solid human tumours and rapidly proliferating non‐neoplastic tissue
Authors: MÜNSTERMANN Ursula , FRITZ Gerhard , SEITZ Gerhard , YIPING Lu , SCHNEIDER Helge R. , ISSINGER Olaf‐Georg
Published: March 2005

Protein kinase CKII (i.e.casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding nonneoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti‐CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII‐specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non‐neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33–350 U/mg protein and in the case of colorectal mucosa 13–106 U mg protein. The amount of CKII determined in the individual tumours was in the range 0.4–1.6 nmol/g tissue.
Characterization and activation of procollagenase from human polymorphonuclear leucocytes
Authors: KNÄUPER Vera , KRÄMER Silke , REINKE Heinz , TSCHECHE Harald
Published: March 2005

Procollagenase of human polymorphonuclear leucocytes was purified to homogeneity using a rapid and reproducible method. The purification procedure included affinity chromatography on zinc chelate Sepharose, ion exchange chromatography on Q‐Sepharose fast flow, followed by affinity chromatography on orange Sepharose and finally a gel‐permeation step on Sephacryl S‐300. It was shown by SDS/PAGE, under reducing conditions, that the latent collagenase of human polymorphonuclear leucocytes consists of a single polypeptide chain with an apparent relative molecular mass of 85000. Upon deglycosylation by endoglycosidase F digestion, the apparent relative molecular mass of the procollagenase was reduced to 53000 which is similar to that of the fibroblast enzyme, and indicates a close relationship between both enzymes. Sequence data were determined by direct automated Edman degradation of the purified polymorphonuclear leucocyte procollagenase. The complete sequence of the propeptide region (residue 1–120) was thereby established. The proteolytic activation of the polymorphonuclear leucocyte procollagenase by various enzymes was investigated by determining the N‐terminal sequences of the intermediate and final activated forms. Activation by chymotrypsin and cathepsin G led to the active form (Mr 64000) by cleaving 79 N‐terminal residues from the proenzyme. Trypsin activates in a two‐step process. Cleavage of 48 N‐terminal residues led to a still latent Mr 70000 species. The final active form (Mr 65000) was obtained by splitting off 20 additional N‐terminal residues.
Three‐dimensional structure of the oligosaccharide terminus of globotriaosylceramide and isoglobotriaosylceramide in solution
Authors: POPPE Leszek , DABROWSKI Janusz , von der LIETH Claus‐Wilhelm , KOIKE Katsuya , OGAWA Tomoya
Published: March 2005

Spatial structures of the oligosaccharide parts of globotriaosylceramide, Gal(α1‐4)Gal(β1‐4)Glc(β1‐1)Cer (Cer = ceramide) and isoglobotriaosylceramide, Gal(α1‐3)Gal(β1‐4)Glc(β1‐1)Cer were investigated in (C2H3)2SO solution by means of laboratory and rotating frame NOE, hydroxyl protons being used as long‐range sensors defining the distance constraints. Both oligosaccharides were found to exist in more than one conformation interconverting rapidly on the NMR time scale. The conformation of the Gal(α1‐4)Gal(β1‐4)Glcβ trisaccharide dissolved in 2H2O appeared to be the same as that of the corresponding part of the glycosphingolipid in (C2H3)2SO solution.
Tissue distribution of avidin and streptavidin injected to mice
Authors: SCHECHTER Bilha , SILBERMAN Rogerio , ARNON Ruth , WILCHEK Meir
Published: March 2005

Radioionated avidin and steptavidin were characterized for their biodistribution and tissue association in Balb/c mice, in comparison to their interaction with cells in vitro. Binding of avidin to spleen and bone‐marrow cells in vitro was up to 20‐fold higher than that of streptavidin, but when tested in vivo avidin clearance from blood and tissues was considerably faster than that of streptavidin. Levels of avidin at 24 h after an intravenous injection were below 1% (of the injected dose/mass tissue) in most organs. Non‐glycosylated avidin was similar in its biodistribution to native avidin. Native streptavidin exhibited higher and prolonged tissue association with 5–10% levels in lung, liver, spleen, kidney and blood, whereas its truncated form showed low tissue levels (1–3%) but a remarkably high affinity to the kidney (80%). Exogenous biotin did not affect streptavidin distribution in vivo but caused a 2–7‐fold increase in the retention of avidin (but not non‐glycodylated avidin) in some of the organs.
In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal α‐d‐mannosidases
Authors: MICHALSKI Jean‐Claude , HAEUW Jean‐François , WIERUSZESKI Jean‐Michel , MONTREUIL Jean , STRECKER Gérard
Published: March 2005

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H‐NMR spectroscopy. The first steps of the degradation involve an (α1‐2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator.

On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2+‐dependent. This process is characterized by the preferential action of an (α1‐3)mannosidase activity, and the formation of Man(α1‐6)Man(α1‐6)Man(β1‐4)GlcNAc and Man(α1‐6)Man(β1‐4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers.

Our results are consistent both with the existence of several specific enzymes for α1‐2, α1‐3 and α1‐6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core α1‐6‐linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (α1‐6)mannosidase form, unaffected in mannosidosis.
A 136‐amino‐acid‐residue COOH‐terminal fragment of colicin A is endowed with ionophoric activity
Authors: BATY Daniel , LAKEY Jeremy , PATTUS Franc , LAZDUNSKI Claude
Published: March 2005

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a ‘STOP‐Shine‐Dalgarno‐sequence‐ATG’ cassette encoding a termination codon, a Shine‐Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137‐amino‐acid‐residue pore‐forming protein designated DA70C comprising the final 136‐amino‐acid‐residue COOH‐terminal of colicin A preceded by an NH2‐terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X‐114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1–3 of the 204‐amino‐acid‐residue colicin pore‐forming domain (containing 10 α‐helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH‐terminal domain.
Limited proteolysis of β2‐microglobulin at Lys‐58 by complement component C1s
Authors: NISSEN Mogens H. , ROEPSTORFF Peter , THIM Lars , DUNBAR Bryan , FOTHERGILL John E.
Published: March 2005

We have now demonstrated that activated complement component C1s cleaves β2‐microglobulin at the position identical to that at which β2‐microglobulin is cleaved in serum of patients suffering from lung cancer. The main cleavage is in the disulphide loop C‐terminal to Lys‐58, generating a modified form of β2‐microglobulin with a two‐chain structure. The C‐terminal Lys‐58 in the A chain is highly susceptible to removal by a carboxypeptidase‐B‐like activity causing the formation of des‐Lys58 ‐β2‐microglobulin. This is the first demonstration of a non‐complement protein substrate for the proteolytic activity of C1s. The C1s‐induced cleavage of β2‐microglobulin can be inhibited in the presence of C1 esterase inhibitor, demonstrating a regulatory function of C1 esterase inhibitor in the C1s‐induced cleavage of β2‐microglobulin.
Strikingly low ATPase activities in flagellar axonemes of a Chlamydomonas mutant missing outer dynein arms
Authors: KAGAMI Osamu , KAMIYA Ritsu
Published: March 2005

The ATPase activities in Chlamydomonas axonemes were compared between wild type and a mutant (oda) that lacks entire outer dynein arms, at various ionic strengths and pH values, and in the presence of different concentrations of high‐molecular‐mass dextran. Over a 0–0.2 M KCl concentration range, the ATPase activity of oda axonemes was found to be 5–12 times lower than that of the wild‐type axonemes. The low activity in oda is surprising since outer arm‐depleted axonemes of sea urchin sperm have been reported to retain about 50% of the normal activity. In both wild type and oda, the ATPase activity of dynein was higher when contained within the axoneme than when released from it with 0.6 M KCl. The ATPase activation within the wild‐type axoneme was inhibited by high ionic strengths or by the presence of dextran. The activation in oda axonemes, on the otherhand, was not inhibited by these factors. These significantly different ATPase properties suggest that the inner and outer dynein arms perform somewhat different functions in this organism.
A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen
Authors: HAREUVENI Mara , TSARFATY Ilan , ZARETSKY Joseph , KOTKES Phillip , HOREV Judith , ZRIHAN Sheila , WEISS Mordechai , GREEN Stephen , LATHE Richard , KEYDAR Iafa , WRESCHNER Daniel H.
Published: March 2005

A monoclonal antibody, H23, that specifically recognizes a breast‐tumor‐associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850‐bp cDNA insert, shows that they are composed of 60‐bp (G + C)‐rich tandem repeating units. The coding strand was determined and codes for a proline‐rich 20‐amino‐acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60‐bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60‐bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragement produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over‐expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.
Characteristics and in vivo occurrence of type VIII collagen
Authors: JANDER Renate , KORSCHING Eberhard , RAUTERBERG Jürgen
Published: March 2005

Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930–3937]. Contaminating type IV collagen was removed by ion‐exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen.
Regulation of mannose 6‐phosphate/insulin‐like growth factor II receptor distribution by activators and inhibitors of protein kinase C
Authors: BRAULKE Thomas , TIPPMER Stefanie , CHAO Huey‐Jiun , von FIGURA Kurt
Published: March 2005

The tumor‐promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6‐phosphate (Man6P) and of insulin‐like growth factor II (IGF‐II) to the Man6P/IGF‐II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1‐oleoyl‐2‐acetyl‐sn‐glycerol, but not with 4α‐phorbol, an inactive tumor‐promoter. The increase is due to a 3–4 fold increase in the number of cell‐surface, receptors, accompanied by a 1.6‐fold increase in ligand‐binding affinity. The internalization rate of the Man6P/IGF‐II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF‐II receptors did not impair the sorting of newly synthesized Man6P‐containing ligands while uptake of these ligands is 2–4‐fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P‐containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF‐I, IGF‐II or epidermal growth factor induces a rapid redistribution of Man6P/IGF‐II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681–686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF‐II receptor redistribution induced by Man6P and growth factors.

These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF‐II receptor cell‐surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.
Guanine nucleotides and Ca2+‐dependent lysosomal secretion in electropermeabilised human platelets
Authors: ATHAYDE C. M. , SCRUTTON M. C.
Published: March 2005

Metabolically stable analogues of GTP, e.g. guanosine 5′‐[γ‐thio]trio]triphosphate (GTP[S]) and guanosine 5′‐[β‐γ‐imido]triphosphate (pp[NH]pG), enhance the extent of Ca2+‐dependent secretion of β‐N‐acetylgluco‐saminidase and β‐galactosidase from electropermeabilised human platelets in the presence of less than 5 μM Ca2+.A similar effect is observed on addition either of 1,2‐dioctanoin or of GTP in the presence or absence of thrombin.

In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30–40 μM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2+‐dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2‐dioctanoin.

The concentration of GTP[S] or pp[NH]pG required to obtain half‐maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5‐hydroxytryptamine, β‐N‐acetylglucosaminidase and β‐galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5‐hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of β‐N‐acetylglucosaminidase and β‐galactosidase.

In the presence of a saturating concentration of GTP[S] marked 5‐hydroxytryptamine and β‐N‐acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of β‐N‐acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C.

Guanosine 5′‐[β‐thio]diphosphate inhibits enhancement of β‐N‐acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone.

Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2+‐dependent platelet lysosomal secretion by activating a guanine‐nucleotide‐binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
Analysis of a soluble mutant des‐methionine interleukin‐2 receptor α chain (Tac protein) produced by transfected mammalian cells
Authors: BECKERS Thomas , HAUSER Hansjörg , HÜSKEN Dieter , ENGELS Joachim W.
Published: March 2005

By using recombinant DNA technology the cytoplasmic and trans‐membrane domain of the human interleukin‐2 receptor α chain (IL‐2Rα, Tac) and of a mutant protein lacking methionine‐residues 18, 25, 44, 88, 92, 126, 149, 167, 205, and 209 (des‐Met IL‐2Rα) encoded by a chemically and enzymatically synthesized gene, were deleted. This leads to secretory expression of soluble wild‐type and des‐Met mutant Tac protein of 42–45 kDa after transfection of BHK‐21 cells. Transfectants secreted up to 1.6 μg soluble wild‐type IL‐2Rα protein/106 cells in 24 h into the culture medium. LTK− cell lines, expressing a large number of wild‐type and des‐Met mutant lowaffinity IL‐2Rα of 50–55 kDa on their surface, shed a truncated form of the Tac protein of about 40 kDa into the culture medium. In contrast to wild‐type IL‐2Rα, shedding of mutant Tac protein is strongly reduced. This phenomenon might be the result of higher protein stability of the mutant receptor which may also explain the about 10 times higher surface expression of des‐Met IL‐2Rα in LTK− cells. There are no significant differences in the biosynthesis and post‐translational modification of mutant or wild‐type Tac proteins either in transfected LTK− or BHK‐21 cells as analysed by pulse/chase labeling experiments.
β‐Galactosidase decreases the binding affinity of the insulin‐like‐growth‐factor‐II/mannose‐6‐phosphate receptor for insulin‐like‐growth‐factor II
Authors: KIESS Wieland , THOMAS Cheryl L. , SKLAR Mark M. , NISSLEY S. Peter
Published: March 2005

The insulin‐like growth‐factor‐II/mannose‐6‐phosphate (IGF‐II/Man6P) receptor binds two classes of ligands, insulin‐like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, β‐galactosidase, to modulate the binding of 125I‐IGF‐II to the receptor. β‐Galactosidase purified from bovine testis was fractionated on a DEAE‐Sephacel ion‐exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I‐IGF‐II to the IGF‐II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125I‐IGF‐II to the receptor. A pool of these fractions from the DEAE‐Sephacel column inhibited 125I‐IGF‐II binding to pure receptor by 80% with the concentration required for half‐maximal inhibition being 25 nM. The inhibition of binding by β‐galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of β‐galactosidase with d‐galactonic acid γ‐lactone did not affect the ability of β‐galactosidase to inhibit the binding of 125I‐IGF‐II to the receptor. Scatchard analysis of IGF‐II binding to pure receptor in the presence and absence of β‐galactosidase showed that β‐galactosidase decreased the binding affinity for IGF‐II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM β‐galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I‐IGF‐II to the IGF‐II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF‐II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF‐II – Sepharose column all exhibited stimulation of 125I‐IGF‐II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I‐IGF‐II binding by Man6P. We conclude that β‐galactosidase decreases the binding affinity of the IGF‐II/Man‐6‐P receptor for IGF‐II by binding with high affinity to the Man6P‐recognition site.