Showing papers in "Carlsberg Research Communications in 1976"

Isolation of carboxypeptidase Y by affinity chromatography

Abstract: Carboxypeptidase Y from bakers’ yeast has been purified in high yields by affinity chromatography. The affinity gel was prepared by coupling the specific inhibitor p-aminobenzylsuccinic acid via an azo linkage to Sepharoseglycyl-tyrosine. This affinity gel was able to bind carboxypeptidase Y specifically and quantitatively from a crude yeast autolysate. The isolated enzyme appeared homogeneous by gel electrophoresis and ultracentrifugation, while isoelectric focusing revealed the presence of two components with isoelectric points of pH 3.56 and 3.66, respectively. Small differences in amino acid composition and enzymatic properties between the enzyme from danish yeast and the corresponding enzyme isolated from Fleichmann yeast suggested the existence of more than one form of this enzyme.

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Abstract: Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases fromBacillus subtilis andBacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of “secondary binding sites”: X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed byKraut and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.

Nutrient uptake in Tetrahymena pyriformis

Abstract: Several morphological structures have been implicated in nutrient uptake in the ciliate protozoon,Tetrahymena pyriformis: food vacuoles, various types of vesicles and the plasma membrane. It is the object of this report to discuss the roles of these organelles in food uptake. Measurements of multiplication rates under conditions where food vacuole formation could be controlled experimentally suggested that the food vacuoles (about 5 μm in diameter) were essential for rapid cell multiplication in various standard growth media. If, however, concentrations of certain specific nutrients (different for different strains ofT. pyriformis) were high, then the cells could multiply rapidly even when food vacuoles were absent. Furthermore, multiplication rates of cells supplied with particulate or dissolved egg albumin as the amino acid source, suggested that the food vacuoles took up particulate egg albumin well, but dissolved egg albumin poorly. The role in food uptake of vesicles with a diameter of less than 1 μm remains largely unknown. Our present knowledge of them is not yet sufficiently detailed to permit estimations of the rates with which they are formed or of their total number per cell. The plasma membrane has carrier-mediated uptake sites for a number of nutrients such as amino acids and nucleosides. It is likely that this type of uptake mechanism plays a quantitatively important role inT. pyriformis whenever such compounds are present in the extracellular fluid.

In vitro synthesis of barley endosperm proteins on wild type and mutant templates

Abstract: Membrane bound and free polyribosomes were isolated from 20 day old barley endosperms. Sucrose gradient analysis revealed distinct polysomal peaks up to heptamers. The isolated polysomes were active in a cell-free protein synthesizing system employing wheat germ extract. SDS-polyacrylamide gel electrophoresis showed that proteins with molecular weights ranging from 200,000 to 10,000 daltons were synthesized. A substantial part of the polypeptides coded for by the template associated with the membrane bound polysomes was identified as hordeins by their solubility in 55% isopropanol and by their co-migration with native hordein on SDS-polyacrylamide gels. Membrane bound endosperm polysomes from a barley mutant defective in hordein synthesis produced in the cell-free protein synthesizing system only a small amount of hordein. Conversely membrane bound polysomes from the endosperm of a mutant giving rise to an increased content of some hordein polypeptides catalyzed a preferential synthesis of these polypeptides in vitro. SDS-polyacrylamide gel electrophoresis revealed that the in vitro template activities of the free polyribosomes from the wild type and mutant endosperms were very similar. The resulting polypeptides had not the solubility characteristics of hordeins.

On the nature of the polymorphism of the small subunit of ribulose-1,5-diphosphate carboxylase in the amphidiploid Nicotiana tabacum

Abstract: The N-terminal amino acid sequence of the small subunit of ribulose-1,5=disphosphate carboxylase from the amphidiploidNicotiana tabacum contains two polymorphisms. From examination of the equivalent sequences in the putative parent speciesNicotiana sylvestris andtomentosiformis it is concluded that the amphidiploidNicotiana tabacum has inherited two alleles for the small subunit of ribulose diphosphate carboxylase, one from each parent species. The alleles continue to be retained and expressed. The relevance of these findings is discussed in relation to the successful adaption of the amphidiploidNicotiana tabacum to a wide range of environments.

Synthesis of δ-aminolevulinic acid by isolated plastids

Abstract: This paper describes a plastid preparation from spinac capable of forming δ-aminolevulinate from α-ketoglutarate. Isolated plastids from immature leaves had this ability while those from mature leaves did not. Radioactivity from α-ketoglutarate-1-14C and α-ketoglutarate-U-14C was incorporated into δ-aminolevulinate to an equal extent. The ability to form δ-aminolevulinate was associated with intact plastids rather than with mitochondria or microbodies. The formation of δ-aminolevulinate was stimulated by light and had a broad pH suptimum from 6.5 to 8.0. The accumulation of δ-aminolevulinate was enhanced by the presence of levulinate, an inhibitor of δ-aminolevulinate dehydratase.

The C and Q banding patterns of the chromosomes of Lilium longiflorum (thunb.)

Abstract: The morphology and the C and Q banding properties of theLilium longiflorum karyotype have been investigated. Only 9 C bands have been observed, while the Q bands occupy 40–50% of the chromosome volume. Some correlation has been observed between regions of higher Feulgen stainability and the Q bands. The very low amount of centromeric C banding may reflect the absence of long stretches of highly repeated DNA. The relationship between the C and Q bands and constitutive heterochromatin is discussed in relation to the premeiotic and somatic interphase inLilium longiflorum.

Properties and morphology of barley embryo acetyl CoA carboxylase

Abstract: Acetyl CoA carboxylase was isolated and purified from barley embryos. The purified enzyme fixed CO2 at the rate of 7 to 7.4 μmoles per min per mg protein. It had a molecular weight of 610 000 daltons and one mole of biotin per mole of enzyme. The purified enzyme aggregates during sepharose 6 B gel filtration. Aggregation of the enzyme can be prevented by using 1% sodium chloride in the elution buffer. The biotin carboxyl carrier protein of the enzyme was identified as a small polypeptide with a molecular weight of 21 000 daltons. This peptide behaves identically to barley chloroplast biotin carboxyl carrier protein during polyacrylamide disc electrophoresis in phenol: acetic acid: urea. The morphology of purified acetyl CoA carboxylase was studied by electron microscopy using a negative staining technique. This enzyme is a globular protein with a size of 275±8 a×192±42 A and displays characteristic cavities.

Polypeptide composition of thylakoids from viridis and xantha mutants in barley

Abstract: The chloroplast membrane proteins from four recessive nuclear gene mutants of barley were studied with sodium dodecyl sulfate gel electrophoresis. The protein patterns of two mutants,xantha-b12 andviridis-zd69, consist of a summation of the polypeptides present in the chloroplast and etioplast membranes of the wild type. The pattern ofviridis-c12, a mutant deficient in photosystem II activity was lacking two bands, one of which is considered to be a component of the reaction center of photosystem II. The pattern ofviridis-k23 lacked three chloroplast specific bands, two of which are components of chlorophyll-protein complex II. The results are discussed in relation to previously established functional and structural characteristics of the mutant plastids.

Composition of epicuticular waxes on barley spikes

Abstract: Hydrocarbons, alkan-1-ol and alkan-2-ol containing esters, β-diketones, aldehydes, primary alcohols, hydroxy-β-diketones, free fatty acids and sometimes secondary alcohols are major lipid classes of epicuticular waxes on the spikes of the three wild type barley varieties Bonus, Foma and Carlsberg-II and many of the 40eceriferum (cer) mutants examined. Division of the spikes into two parts, the awns and the spike minus awns, before wax isolation reveals that (i) β-diketones, hydroxy-β-diketones and esterified alkan-2-ols occur only on the spike minus awns, (ii) secondary alcohols are present only on the awns of Foma and its mutants and (iii) the other lipid classes are found on both parts of the spike. The chain length distributions of the lipid classes common to both parts of the spike are in some cases similar and in others different.

The influence of cations and methylamine on structure and function of thylakoid membranes from barley chloroplasts

Abstract: The effects of cations on the structure and activity of isolated barley (Hordeum vulgare cv. Svalofs Bonus) chloroplast lamellar systems were studied. Three separate effects of cations were recognizable. (1) Cations in high concentrations (in excess of 100–200 mM NaCl) are required to maintain granal stacks once the outer chloroplast envelope is broken. In 30 mM NaCl the majority of the lamellar systems exist as well-separated thylakoids, even in the presence of high concentrations of sucrose (330 and 660 mM). The lamellar systems photoreduce ferricyanide at coupled rates whether they are in the granal or non-granal configuration. (2) Cations are required for Hill reaction activity. This requirement becomes apparent following loss of thylakoid membrane integrity at very low cation concentrations. Maximum activation of the photoreduction of ferricyanide occurs at 30 mM NaCl, and divalent ions (Mg++, Ca++, Mn++) are 12 times more effective than monovalent ions (Na+, K+). (3) Cations are necessary to preserve the integrity of thylakoid membranes. Below about 8 mM NaCl, the thylakoids swell and this change is accompanied by progressive loss of Hill activity and the capacity to maintain a light-dependent proton gradient. Divalent cations are more than 200 times as effective as monovalent ions in preventing these changes. Hill reaction activity, but not proton pump activity, is regained by adding cations back to the swollen thylakoids (cation effect 2, above) and the new rate of Hill reaction activity is the same as in chloroplasts uncoupled with methylamine. The re-establishment of Hill reaction activity is accompanied by appression of the swollen thylakoids. The uncoupler methylamine causes stacking of thylakoids and collapsing of intrathylakoidal spaces.

Ribulose-1,5-diphosphate carboxylase from barley (Hordeum vulgare)

Abstract: This paper presents a simple and rapid procedure for the isolation of ribulose-1,5-diphosphate carboxylase from leaves. By column chromatography on Sephadex G-25 and Sepharose 6B combined with concentration by ultrafiltration high yields of undenatured protein are obtainable in less than one working day. RudP carboxylase purified from barley in this way has been characterized. The molecular size is similar to that of spinach, wheat and oat. The apparent molecular weight determined by column chromatography was found to be 510,000, approximately 3% higher than that for the tobacco protein in the same systems. RudP carboxylase from barley consists of two different kinds of subunits with the same molecular weight properties as described for other plants. The amino acid composition of the native protein shows similarities with another monocotyledon, oat, both having lower contents of leucine and tyrosine than the dicotyledons, spinach and tobacco. The high content of tryptophan in barley RudP carboxylase gives a higher extinction at 280 nm than has been reported for other organisms (E 280 nm 1‰ =2.06). This paper also describes a mapping technique for the tryptic peptides of the subunits of RudQ carboxylase by two-dimensional high voltage paper electrophoresis which is rapid, reproducible and gives well defined spots. The peptide mapping technique is well suited as a screening method for RudP carboxylase mutants.

Gel filtration chromatography of oligosaccharides

Abstract: Gel filtration chromatography on Bio-Gel P-4 at 65°C has been used to separate oligosaccharides obtained from amylose, dextran, pullulan, and starch. The position of each oligosaccharide in the elution profile has been expressed by its Kav value. Each series of homologous oligosaccharides (e. g. DP2-15/DP3-27) gives a straight line in a plot of-log Kav versus DP. Comparison of isomeric oligosaccharides with α-1,4 and/or α-1,6 glucosidic linkages in a defined linear sequence reveals characteristic differences, probably related to the size (»hydrodynamic volume«) of these linear oligosaccharides (α-1,6>α-1,4: α-1,6>α-1,4). In contrast, isomeric branched oligosaccharides have almost identical Kav values close to that of the corresponding malto-oligosaccharide (α-1,4: α-1,6 ≈α-1,4 only). This permits quantitative gel filtration chromatography according to DP of acid and enzymic hydrolyzates of starch. Cyclic, α-1,4 linked oligosaccharides (Schardinger dextrins) appear in size to be considerably smaller than the corresponding malto-oligosaccharides. The results show that although oligosaccharides are eluted in approximately the reverse order of their molecular weight fine structure of the oligosaccharide seems to determine its position in the elution profile.

Dual capacity for nutrient uptake in tetrahymena importance of the oral uptake system for Fe and Cu uptake

Abstract: We have reported that a mutant ofTetrahymena pyriformis with heat-sensitive development of the oral apparatus can be grown indefinitely without food vacuoles if the medium is supplemented with folinic acid and a mixture of trace metal salts. We report here that the trace metal mixture can be replaced completely and specifically by salts of iron and copper. Fe(II) and Fe(III) are interchangeable. Addition of citrate has proven useful to reduce precipitate formation and improve the reproducibility of growth of the mutant cell. Thus it appears to serve as an Fe buffer. From the increased concentrations of Fe and Cu required to permit good growth of the mutant strain at 37°C, we conclude that the oral uptake system plays a much more important role in the case of these two metals than the surface uptake system. The oral uptake system may facilitate Fe uptake in at least two ways: a) by a mechanical concentration of precipitates affected by the ciliary membranelles surrounding the oral cavity and, b) by lowering of the pH of the food vacuole and thereby releasing Fe from precipitates and from complexes which cannot be transported across the membrane as such. The second factor may also be important in Cu uptake. A specific effect of Mg and Fe uptake or retention in the mutant strain growing without food vacuoles has been detected. The significance of this effect remains unclear. Practical implications of the findings are discussed.

Enzymatically active, cross-linked pig heart lactate dehydrogenase crystals

Abstract: Pig heart lactate dehydrogenase crystals were cross-linked with octanediimidic acid dimethylester. The resulting cross-linked crystals were insolable in dilute phosphate buffer and had retained a high specific activity. Amino acid analyses showed that about twelve lysine residues out of twenty-three per subunit had been chemically modified. The cross-linked crystals showed increased stability in urea compared to the soluble enzyme. The differences caused by the cross-linking process to the properties of the enzyme are briefly discussed.

Polypeptide composition of internal membranes from barley etioplasts

Abstract: The polypeptides of isolated etioplast membranes and chloroplast membranes were solubilized in sodium dodecyl sulfate. Protein band patterns were obtained by electrophoresis on polyacrylamide slab gels with separating gels containing either a uniform polyacrylamide concentration or a concentration gradient. Several characteristic chloroplast bands were absent or reduced in intensity in patterns of etioplast membranes. Conversely, certain bands found in etioplast membrane patterns were reduced or absent in patterns of chloroplast membranes. Among these was a distinct doublet detected only in patterns of etioplast membrane polypeptides obtained with gels lacking urea. Some bands, for example those tentatively identified as subunits of coupling factor were found in patterns of etioplast as well as chloroplast membranes.

Large scale isolation and partial characterization of some carboxypeptidases from malted barley

Abstract: The carboxypeptidase activity in the water extract of malted barley was resolved into five components; two of these (CP 1-1 and CP 2-1) were obtained in high purity and were partially characterized in regard to enzymatic and chemical properties. Both appeared homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The MW of CP 1-1 was 81,500 by sedimentation equilibrium and 96,000 by gel filtration; for CP 2-1, the values were 85,000 and 94,000 respectively. Sedimentation coefficients, S20,w, were 5.5 for CP 1-1 and 5.6 for CP 2-1. The isoelectric point was pH 5.75 for both enzymes. Carbohydrate comprised 6.6% of CP 1-1 and 7.6% of CP 2-1. The carboxypeptidases hydrolyzed Z-Phe-Ala, Z-Phe-Ser, Z-Phe-Leu, Z-Glu-Tyr and ATEE, plus other substrates; the pH of optimal activity and Km values were determined for several of these. Carboxypeptidase activity was inhibited strongly by diisopropylfluorophosphate andp-hydroxymercuribenzoate, mildly by a number of carboxylic acids and their derivatives, and weakly by metal chelators, metal ions and urea.

Isolation of human carbonic anhydrase B and C and apocarbonic anhydrase by affinity chromatography

Abstract: Human carbonic anhydrase B and C have been purified in high yields by affinity chromatography, using two different affinity gels, incorporating the potent benzenesulfonamide inhibitor, but coupled to Sepharose through different «spacer-arms». Although both affinity gels bind carbonic anhydrase B and C quantitatively, they exhibit different binding capacities. Sepharose-glycyl-L-tyrosine-azobenzenesulfonamide was used to separate the mixture of isoenzymes from the bulk hemoglobin in the hemolysate of human erythrocytes. This affinity gel had a capacity of 15 mg of enzyme pr. ml packed gel. The isoenzymes were eluted with thiocyanate an inhibitor of carbonic anhydrase.

The cell cycle in heat- and selection-synchronized schizosaccharomyces pombe

Abstract: Schizosaccharomyces pombe, growing in a complex medium at 32°C, was treated with 6 or 7 heat shocks (30 min, 41°C) spaced one normal cell generation apart (110 min, 32°C). This treatment results in synchronization of cell division, nuclear division, and DNA synthesis. Between successive shocks increases in the activities of ATCase and OTCase are stepwise. However, steps are not seen at constant temperature, neither in free running heat synchronized, nor in selection synchronized cells. The enzyme steps we have studied therefore seem to be directly induced by the temperature shocks, and they are dissociable from the pattern of classic cell cycle events, which we find almost identical in heat and in selection synchronized cells.

Ribulose 1,5-diphosphate carboxylase from oenothera. purification and a peptide mapping procedure for the subunits

Abstract: Ribulose 1,5-diphosphate carboxylase has been purified from fully expanded leaves ofOenothera. The isolation procedure overcame the problems which resulted from a high content of mucilage and phenolic compounds in the leaves. Protein extraction into dilute buffer containing a phenol adsorbant and reducing agents followed by a combination of gel filtration and ion exchange chromatography allowed the enzyme to be essentially purified to homogeneity. The purified RuDP carboxylase had a specific activity of 1.5 μmoles CO2 fixed. min−1. mg−1 at pH 7.8 and 25°. The two subunits could be dissociated in detergent solutions and were found to have molecular weights and amino acid compositions similar to the respective subunits from other sources. A two-dimensional mapping procedure employing ion exchange and paper chromatography was used to partially characterize the tryptic peptides of the RuDP carboxylase subunits; this technique may be useful in interspecific comparisons of the primary structure of the two subunits fromOenothera species.

Studies on the heavy spherical (refractive) bodies of freshwater Amoebae. I. Morphology and regeneration of HSBS in Chaos carolinense

Abstract: The heavy spherical bodies ofAmoeba andChaos are refractive organelles, varying in size from ca. 10 μm in diameter, down to the limit of resolution of the light microscope. These inclusions have many characteristics in common with the phosphate-rich, «volutin» granules of other unicellular organisms, but differ from the latter in being constantly present in healthy growing amoebae, while in other organisms they appear only under conditions of nutritional imbalance. The fine structure of the HSBs ofAmoeba andChaos show that the organelles are membrane-bound, with an electron translucent periphery, and an electron dense inner core which sublimes in the electron beam, but which is stabilized by lead staining. During centrifugation in vivo, the HSBs collect at the centrifugal pole of the amoebae; under favourable conditions, a HSB-sack is formed at the heavy pole, and can be excised, leaving amoebae, normal in other respects, but containing only ca. 5% of the normal complement of HSBs. Using this method for obtaining practically HSB-free amoebae, the regeneration of HSBs was studied inChaos carolinense, by determining the number and size distribution of HSBs in a standard volume in fixed, whole mounts by phase contrast microscopy. It was found that although the number of HSBs per standard volume in both fed and starved amoebae returned to that found in control, unoperated,Chaos by 7 days after operation, the size distribution did not return to normal until ca. 50 days after excision of the HSBs, indicating that HSBs originate from precursors in the cytoplasm. Operated amoebae showed normal feeding and locomotory behaviour, and their division rate was similar to that of control amoebae. However, HSB-free amoebae were more prone to rupture at an air-water interface, presumably owing to their lower specific gravity.

The hydrolysis of the three clupein components by subtilisin Novo, subtilisin Carlsberg and nitrated subtilisin Carlsberg

Abstract: The three components of clupein (YI, YII and Z) were hydrolyzed by subtilisin Carlsberg and subtilisin Novo. Subtilisin Novo hydrolyzed all three components much faster than did subtilisin Carlsberg, but components YII and Z were hydrolyzed with the same initial rate and faster than component YI. The primary bonds cleaved in component YII were Ser22-Arg23 followed by Ala8-Ser9. Ser21-Arg22 followed by Ala9-Ser10 were the primary bonds cleaved in clupein-Z. It was further demonstrated that nitrated subtilisin Carlsberg had become more specific for cleavage of the Ser-Arg bond compared with the unmodified enzyme. With clupein YI as the substrate, the peptide pattern obtained was too complicated for a particular bond to be identified as the primary attacking point for the two unmodified enzymes. Nitrated subtilisin Carlsberg attacked initially the bonds Arg5-Ser6 and Ser6-Ser7.

An overhead cuvette stirrer for studying enzymes attached to microgranular matrices

Abstract: An overhead stirrer was constructed to maintain a homogeneous suspension, in a spectrophotometer cuvette, of microgranular celluloses used for enzyme immobilisation. The presence of limited amounts of the celluloses was shown to have no effect on the spectra of solutes.

Buoyant titration of bovine mercaptalbumin chemically modified at the carboxyl groups

Abstract: Bovine mercaptalbumin has been modified at the carboxyl groups by means of 1-ethyl-3 (3-dimethylisopropyl) carbodiimide and glycine amide. A stable derivative was obtained after modification of 54 groups. The modified protein differed only slightly from the unmodified one with respect to the optical rotatory dispersion parameters ao, bo and χc. This preparation was studied in the analytical ultracentrifuge in a CsCl gradient in the pH-interval from 2 to 11. The buoyant densities were determined and compared with those of the unmodified protein in the same pH-interval. The contribution from the carboxyl groups to the buoyant density is discussed and compared with results obtained with other proteins and synthetic polypeptides.

One hundred years of originality, quality and style

Abstract: My choice of a title for this short lecture on the Carlsberg Laboratory and its impact over the years was made after a lot of thought about why some centers of science become international Meccas and so many others never quite make it. Thousands of scientists ponder the problem of where they might best go to attain professional breadth and skill in their specialty. It is generally fairly easy to give advice to such individuals. At any given time, certain scientific centers stand out in everyone's mind: the MRC lab at Cambridge, with its glittering constellation of stars, the Pasteur Institute in the exciting and fermenting days of JACOB and MONOD at their best, OTTO WARBURG'S laboratory in the twenties and thirties when the machinery of metabolism first began to be studied in detail, and my own institution, the NIH, during the days of the Vietnam doctor draft when we were able to help a large group of bright, young MD's to stay out of a senseless war. To protein chemists (omitting of course X-ray crystallographers who travelled elsewhere), the name of the Carlsberg Laboratory has for many years been synonymous with the initiation, prediction or implementation of research in this particular field. It also happens to be in Copenhagen, which gives an initial advantage. In the days of the first director, JOHAN KJELDAHL, I suppose this did not really apply. International travel grants had not been invented and the level of science in the Western Hemisphere and in Asia had not yet benefited from the individuals and ideas that were subsequently to come from Europe and Great Britain. However, from the time of SORENSEN to the present, almost every protein-oriented laboratory in the world has been flavored with the scientific spices that itinerant students brought home from Copenhagen. As I will try to illustrate to you shortly, using examples from my own scientific career, the conscious or subconscious choice made in research direction or emphasis has frequently been the product of the Carlsberg Laboratory's intellectual flavor. Let me first say a few things about the history of