bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protease Resistance and Conformation of Laminin
Authors: OTT Ursula , ODERMATT Erich , ENGEL Jürgeii , FURTHMAYR Heinz , TIMPL Rupert 
Published: March 2005

Laminin, obtained from a tumor basement membrane, was treated with neutral proteases (trypsin, chymotrypsin, elastase, subtilisin, Stuphylococcus aureus protease), which all produced similar fragment patterns upon prolonged digestion. The patterns were different from those obtained for fibronectin, which showed a much higher susceptibility to proteolysis by the same enzymes. Four large fragments could be purified which accounted together for more than half of the mass of laminin. They were found to differ in size, amino acid composition, spectral properties and antigenicity. The largest fragment 1 (Mr 260000‐300000) was rich in half‐cystine (120 residues/1000) and showed a circular dichroism spectrum indicating the absence of α helix and β structure. Fragment 3 (Mr= 50000) possessed beta; structure and was able to bind onto heparin‐Sepharose. Fragments 2 (Mr= 50000) and 4 (Mr= 75000) were related structures and their relative yields depended on the protease used. They showed spectra similar to those of fragment 1. Electron microscopy revealed that fragment 1 consists of three rodlike elements (length 26 nm) connected to each other at one end. The other fragments appeared as globules (fragment 3), short rods (fragment 2) or globules connected to a short rod (fragment 4). Data obtained from limited proteolysis indicated that fragment 1 and 4 (or 2) are in close proximity to each other in the three short arms of laminin, which in its intact form has the shape of an asymmetric cross. The long arm appeared to be readily degraded by proteases.Circular dichroism studies of native laminin indicated about 55%, aperiodic structures, 15%β structure and 30%× helix. The α helix was readily destroyed by proteolysis and showed a sharp, reversible transition at 58° C. Stability of these structures was decreased by reduction of disulfide bonds or by increasing concentrations of guanidine. Heat denaturation rendered laminin susceptible to plasmin, which did not degrade the native protein. Cleavage occurred mainly within the 440000‐Mrpolypeptide chain of laminin and was accompanied by a partial loss of the long arm.
Glucose Transport in Salmonella typhimurium and Escherichia coli
Authors: POSTMA Pieter W. , VAN REE Ronald , NEYSSEL Oense M. 
Published: March 2005

We have investigated the claim by Schweiger and coworkers [Eur. J. Biochem. I02 (1979) 231 ‐236] that glucose transport in Escherichia coli is catalyzed mainly by an ATP‐dependent transport system instead of the phosphoenolpyruvate : sugar phosphotransferase system. A major argument was the differential effect of 2.4‐dinitrophenol on glucose uptake and the transport of its non‐metabolizable analogue, methyl α‐glucoside. Whereas the first was inhibited, the second was stimulated. When subsequent glucose metabolism is prevented by introducing mutations that eliminate glucose 6‐phosphate metabolism, 2,4‐dinitrophenol does not inhibit glucose transport. Although dinitrophenol inhibited in wild‐type cells of E. coli and Salmonella typhimurium the uptake of I4C label in cells using [U‐14C]glucose as a substrate, disappearance of glucose from the medium was not affected or only slightly affected. Since uptake represents a combination of transport and subsequent metabolism, retention of labelled material depends on the balance of incorporation of label in cellular material and efflux of labelled compounds. Our experiments show that inhibition of the uptake of labelled glucose by 2,4‐dinitrophenol is not due to inhibition of transport as suggested by Schweiger and coworkers, but to increased efflux of labelled compounds such as acetate and pyruvate. In addition, incorporation of label in cellular material is lowered by dinitrophenol. Inhibition of uptake by dinitrophenol is found with many labelled sugars, including mannitol, galactose and glycerol, the transport of which is energized in quite different ways.We conclude that there is no need to postulate a novel ATP‐driven system for glucose transport. All results can be explained with the phosphoenolpyruvate : glucose phosphotransferase system as the main if not sole glucose transport system.
Characterization of the Active Site of Ribonucleotide Reductase of Escherichia coli, Bacteriophage T4 and Mammalian Cells by Inhibition Studies with Hydroxyurea Analogues
Authors: KJØLLER LARSEN Ingrid , SJÖOBERG Britt‐Marie , THELANDER Lars 
Published: March 2005

Hydroxyurea specifically inhibits the enzyme ribonucleotide reductase by inactivating the essential tyrosine free radical in one of the two non‐identical subunits constituting the enzyme. Studies on the reactivity of hydroxyurea analogues towards the free radical salt, potassium nitrosodisulphonate, and the radical‐containing B2 subunit of Escherichia coli ribonucleotide reductase showed that, to obtain maximal enzyme inhibition, the most important parameter of an analogue was the ability to undergo a one‐electron oxidation. Also, a common geometrical feature for the analogues with high inhibitory potency was the planarity of the molecules.Results of inhibition studies with analogues containing bulky substituents but having about the same ability to reduce the free radical salt suggested that the tyrosine free radical of protein B2 is located in a pocket about 0.4 nm wide and more than 0.6 nm deepThe ribonucleotide reductases of E. coli, T‐4infected E. coli and mammalian cells all contain the same tyrosine free radical structure. Still the mammalian reductase showed a 75‐fold higher sensitivity to inhibition by 3,4‐dihydroxybenzohydroxamic acid than the E. coli reductase. In contrast, the sensitivity to hydroxyurea was the same for both enzymes. The dihydroxybenzohydroxamic acid and hydroxyurea both reacted immediately (t½ > 5) with the free radical salt. Therefore the difference in sensitivity between the mammalian and bac‐ terial reductase most probably reflects different topologies of their active sites with the site of the mammalian enzyme being more exposed.The T4‐induced reductase showed a 10‐fold increased sensitivity towards both hydroxyurea and the poly‐ hydroxybenzohydroxamic acids compared to the E. coli enzyme, indicating a greater ability of the T4 tyrosine free radical to undergo a one‐electron reduction. A closer understanding of the different topologies of the active sites of different ribonucleotide reductases could be of great value in the design of target‐directed drugs.
Dolichyl‐Phosphate Phosphatase and Dolichyl‐Diphosphate Phosphatase in Rat‐Liver Microsomes
Authors: BELOCOPITOW Enrique , BOSCOBOINIK Daniel 
Published: March 2005

Dolichyl‐phosphate phosphatase and dolichyl‐diphosphate phosphatase activities of a liver‐cell microsomal preparation were solubilized by treatment with Triton X‐100. The I00000 × g supernatant was then passed through a column of Sepharose‐4B – concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6‐phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospho‐ lipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X‐100 for the two enzymes. Dolichyl‐phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl‐diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi.Dolichyl‐diphosphate phosphatase was more inhibited by divalent cations than dolichyl‐phosphate phos‐ phatase. The apparent Km of dolichyl‐phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl‐phosphate phosphatase, but it produced a stronger inhibition on dolichyl‐diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.
Kinetic and Structural Differences between Cytochrome c Oxidases from Beef Liver and Heart
Authors: MERLE Peter , KADENBACH Bernhard 
Published: March 2005

The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eight‐ fold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content.
The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c‐depleted membranes, deoxycholate‐dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low‐affinity and for the high‐affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes.
Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme.
By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase iso‐ enzymes in liver and heart is discussed.
Infrared Spectroscopy, a Tool for Probing Bacterial Peptidoglycan
Authors: NAUMANN Dieter , BARNICKEL Gerhard , BRADACZEK Hans , LABISCHINSKI Harald , GIESBRECHT Peter 
Published: March 2005

Infrared spectroscopic measurements are used to obtain insights into the three‐dimensional architecture of peptidoglycan (murein), the rigid component of almost all bacterial cell walls. The infrared spectra of various types of peptidoglycans (including all chemotypes and examples of the so called A and B groups) were compared to each other and to those obtained from crystalline chitin.All peptidoglycans investigated exhibited very similar infrared spectra. In particular the conformationally sensitive amide A, I and I1 absorption bands were found to be constantly centered around 3300 cm−1, 1657 cm−1 and 1534 cm−1 respectively; furthermore, the spectral region between 1200 cm−1 and 800 cm−1, characterized by several strong absorption bands connected to complex sugar ring modes, proved to be remarkably uniform. Additionally the infrared spectra remained significantly constant between −175 °C and +75 °C and turned out to be rather independent of sample preparation (solvent replacement, freeze‐drying and film producing).An analysis of band half‐widths revealed no high crystalline state of order of peptidoglycan. On the basis of band positions and half‐widths of amide bands, regular conformations like α helices of β pleated sheets could be excluded.Several distinctive, fingerprint‐like spectral features of the various murein samples permitted a facile identification of individual peptidoglycans. Moreover, infrared spectroscopy seems to be very promising as an analytical tool, e.g. for tracing variations of cell wall structure, detecting conformational changes and estimating crosslinking indices in a quick and simple way.The comparative analysis of amide band positions and band half‐widths yielded substantial differences between infrared spectra of chitin and murein, thus rejecting previous models based on the assumption of a nearly crystalline chitin‐like structure of the glycan chains of murein.
Radioactive Human Lymphoblastoid Interferon
Authors: YONEHARA Shin 
Published: March 2005

Human lymphoblastoid interferon (α type), labeled with [3H]leucine added to virus‐induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate.Pulse‐labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period.Competitive radioimmunoassay for human interferon a was achieved by the use of purified radioactive inter‐ feron, anti‐(interferon α) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon a, and 100 international reference units of interferon a could be measured in this way.
Classification of Trimethoprim‐Resistant Dihydrofolate Reductases Mediated by R‐Plasmids Using Isoelectric Focussing
Authors: BROAD David F. , SMITH John T. 
Published: March 2005

An analytical isoelectric focussing technique has been developed to distinguish between different groups of R‐plasmid‐mediated trimethoprim‐resistant dihydrofolate reductases. Those mediated by the R‐plasmids R483 and R721, which are type I dihydrofolate reductases have isoelectric point (PI) values of 6.4 whereas those mediated by the R‐plasmids R67bis and R27, which are type I1 enzymes, have PI values of 5.5. There has been some confusion as to the classification of the dihydrofolate reductases mediated by R‐plasmids R388 and R751; however, our studies indicate that the one mediated by R388 is a type I1 enzyme, having a PI value of 5.5, whereas the one mediated by R751 is different from both the type I and type I1 enzymes having a PI value of 7.2. The differences in PI value of these three groups of trimethoprim‐resistant enzymes were confirmed by DEAE‐ cellulose chromatography at pH 8.5 where it was found that the type I enzymes were eluted with a different concentration of NaCl than were the type I1 enzymes including that mediated by R388, whereas the enzyme mediated by R751 did not bind to the DEAE‐cellulose at this pH.A ‘scale‐down’ technique was developed for analytical isoelectric focussing and was used to study the dihydrofolate reductases mediated by ten R‐plasmids present in trimethoprim‐resistant Escherichia coli strains isolated from pigs. All these R‐plasmids coded for type I enzymes and all also coded for resistance to streptomycin, indicating that the trimethoprim‐resistance gene(s) were present on a transposon similar to Tn7.
Tuftsin Analogs for Probing Its Specific Receptor Site on Phagocytic Cells
Authors: GOTTLIEB Philip , BERETZ Alain , FRIDKIN Mati 
Published: March 2005

Six new analogs of the phagocytosis‐stimulating peptide tuftsin were synthesized with the eventual aim of characterizing and isolating the tuftsin receptor. These analogs can be classified as follows : (a) photoaffinity label‐ ling analogs for the specific covalent attachment to the tuftsin receptor; (b) fluorescent analogs containing either rhodamine or dansyl fluorescent probes for microscopic visualization of the tuftsin receptor; (c) biotin analog for separation and purification of the receptor by affinity methods.In this paper we describe the various synthetic pathways employed to introduce sensitive prosthetic groups into the tuftsin molecule while preserving its biological activity. Activities of the various analogs synthesized as compared to tuftsin in biological and receptor‐binding assays are described. All analogs are able to stimulate phagocytosis of the macrophage cell as well as compete specifically for tuftsin binding sites on these cells.
Two Forms of Adrenodoxin Reductase from Mitochondria of Bovine Adrenal Cortex
Authors: SUHARA Katsuko , NAKAYAMA Kazuo , KATAGIRI Masayuki , TAKIKAWA Osamu 
Published: March 2005

Two forms of adrenodoxin reductase, differing in isoelectric properties and thermal stability, have been highly purified from bovine adrenal cortex mitochondria. Both preparations appear to be homogeneous based upon polyacrylamide disc‐gel electrophoresis, isoelectric focusing, and ion‐exchange chromatography. Both are monomeric proteins containing one FAD per molecule (Mr 50000) and exhibit spectra typical of flavoproteins. The two preparations are indistinguishable by their amino acid compositions and N‐terminal (serine) and C‐ter‐ minal (leucine) amino acids, but reveal distinct sugar contents. Electrophoretic mapping of the protease digests of the two preparations indicates that the two have identical, or closely similar, primary structures.Each form functions equally as an adrenodoxin‐dependent electron transport component from NADPH to the P‐450 cytochromes in the two different reconstituted steroid monooxygenase systems which catalyze cholesterol side‐chain cleavage reaction and steroid 11β‐(18‐)hydroxylase reaction, respectively. In all adrenodoxin reductase‐ containing fractions from adrenal cortex so far examined, i.e. the soluble fraction, and the sonic‐solubilized and detergent‐solubilized mitochondrial fractions, a constant ratio of about 2:5 between the two forms is found as judged by the DEAE‐cellulose chromatogram. No evidence has been obtained for mutual conversion of the two forms of the reductase, or for dissociation of the sugar moiety during the purification process and under various conditions examined.
Purification of l‐Glutamate‐Dependent Citrate Lyase from Clostridum sphenoides and Electron Microscopic Analysis of Citrate Lyase Isolated from Rhodopseudomonas gelatinosa, Streptococcus dacetilactis and C. sphenoides
Authors: ANTRANIKIAN Garabed , KLINNER Christina , KÜMMEL Angela , SCHWANITZ Diethard , ZIMMERMANN Thomas , MAYER Frank , GOTTSCHALK Gerhard 
Published: March 2005

Citrate lyase from Clostridium sphenoides was purified 72‐fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of l‐glutamate. The purified enzyme was only stable in the presence of 150 mM l‐glutamate or 7 mM l‐glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the l‐glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular l‐glutamate concentration.Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: ‘star’ form, ‘ring’ form and ‘triangle’ form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of l‐glutamate.
The Alkylating Agent, Dimethyl Sulphate, Stimulates ADP‐Ribosylation of Histone H1 and Other Proteins in Permeabilised Mouse Lymphoma (L1210) Cells
Authors: THI MAN Nguyen , SHALL Sydney 
Published: March 2005

Exposure of mouse lymphoma L1210 cells to the alkylating agent, dimethyl sulphate at 500 μM for 1 h produced an eightfold increase in the activity of nuclear ADP‐ribose transferase as measured by [3H]NAD incorporation after permeabilisation of the cells. The radioactive product formed was shown to be (ADP‐ribose)n, (a) by identification of the snake venom phosphodiesterase products as 5′AMP and phosphoribosyl‐AMP,(b) by its hydrolysis by snake venom phosphodiesterase and poly(ADP‐ribose) glycohydrolase but not by DNase and RNase, and (c) by treatment with the ADP‐ribosyl transferase inhibitor, 3‐aminobenzamide, which almost completely prevented [3H]NAD incorporation.Of this acid‐insoluble radioactivity, 4% was associated with the histone fraction while over 80% was extracted with the non‐histone chromatin protein fraction. After treatment with dimethyl sulphate, radioactivity in the histone fraction increased 20‐fold; the radioactivity in this histone fraction increased proportionately from 4% to 10% of the total radioactivity incorporated.Analysis of the histones on acid/urea gels revealed that most of the [3H]NAD incorporated co‐migrated with histone H1, both with and without dimethyl sulphate treatment, and there was a large increase in radioactivity of histone H1 after this treatment. The average chain length of newly synthesised (ADP‐ribose)n on histones was between three and four ADP‐ribose units.The nature of the ADP‐ribosyl–histone bond was investigated; neutral hydroxylamine liberated 56% of the (ADP‐ribose)n, a further 30% was released by alkaline hydrolysis and 14% was resistant to alkaline hydrolysis.This stimulation of the ADP‐ribosylation of histones by an alkylating agent known to damage DNA provides further support for the participation of ADP‐ribosylation of chromatin proteins in DNA repair.
A New Acrosin Inhibitor from Boar Spermatozoa
Authors: TSCHESCHE Harald , WITTIG Bernd , DECKER Günther , MÜLLER‐ESTERL Werner , FRITZ Hans 
Published: March 2005

A new proteinase inhibitor (Mr 7500) was isolated to apparent homogeneity from boar spermatozoa by repeated gel filtration on Sephadex G‐50 and affinity chromatography on concanavalin‐A‐Sepharose 4B. The inhibitor strongly inhibits boar acrosin in a competitive 1:1 stoichiometric reaction with a Kass= 7 × 1010l mol−1.The inhibitor is a glycoprotein and represents a first member of a new class of proteinase inhibitor with a rather short polypeptide backbone of only 42 amino acid residues and a low cystine content. The basic protein (isoelectric point 9.4) contains a single disulfide loop, which is easily reducible by sodium borohydride. Upon reduction the inhibitory activity is lost, but rapidly regained after air reoxidation of the corresponding half‐cystine residues. The reactive site residue was established to be arginine by inhibition with 2,3‐butanedione. The inhibitor is rather specific for acrosin and inhibits bovine trypsin only to a limited extent. However, incubation with catalytic amounts of trypsin (or acrosin) at acid pH (pH 2–3) rapidly leads to a limited proteolysis at the reactive site with formation of 67% modified (reactive site hydrolysed), but still active inhibitor. The equilibrium constant was established to be Khyd= 2.0.