Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Sterol conjugates of two phenotypically different calli of Beta vulgaris

Steryl glycosides are the predominant form of sterol at 88% of the total sterol in non-betalain producing calli of Beta vulgaris. The total sterol decreases and sterol form shifts from steryl glycosides to 97% free sterol upon the transition of non-betalain to betalain producing calli. A substantial decrease in stigmasterol (24--ethylcholesta-5,22E-dien-3-ol) and sitosterol (24-ethylcholest-5-en-3-ol) levels is observed during this transition, and alters the ratio of ⁷:⁵ sterols. Spinasterol (24- ethyl-5-cholesta-7,22E-dien-3-ol) is the dominant sterol at 43% and 95% of the total sterol in non-betalain producing and betalain producing calli. The level of 22-dihydrospinasterol (24-ethyl-5-cholest-7-en-3-ol) is reduced in both calli to 3% from 25% in leaves. Lanosterol (4,4,14-trimethyl-cholesta-8(9),24-dien-3-ol) and cycloartenol (9,19-cyclopropyl-4,4,14-trimethyl-cholest-24-en-3-ol) were identified in betalain and nonbetalain producing callus respectively.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Polyvalent GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (T <Subscript>α</Subscript>) as the most potent recognition factors involved in <Emphasis Type="Italic">Maclura pomifera</Emphasis> agglutinin–glycan interactions

Summary The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Gal13GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin–glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAc1Ser/Thr (Tn) and Gal13GalNAc1Ser/Thr (T) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 × 10⁵, 4.6 × 10⁴ and 3.9 × 10⁴ more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a⁺) related disaccharide (GalNAc14Gal) and P^k/P₁ active disaccharide (Gal14Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA–glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T (M.W. > 4.0 × 10⁴) >> Tn-containing glycopeptides (M.W. < 3.0 × 10³) > monomeric T/Tn and P (GalNAc13Gal) > GalNAc > Gal >> Man, LAra, DFuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.     

The microbial production of 3aα-H-4α- (3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-indan-1-one-δ-lactone from cholesterol

Summary A mutant strain of Rhodococcus australis CSIR-236.457 which accumulates 3a-H-4-(3-propionic acid)-5-hydroxy-7a-methylhexahydro-indan-1-one--lactone from cholesterol, stigmasterol and -sitosterol was studied. The product is produced in a 60% molar yield in a dilute black strap molasses medium containing 6–12g/l cholesterol after a 72 hour fermentation period.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Increase of catalytic activity of α-chymotrypsin in organic solvent by co-lyophilization with cyclodextrins

-Chymotrypsin was lyophilized in the presence of 2,3,6-tri-O-methyl -cyclodextrin, and it displayed activity 40 fold higher than free -chymotrypsin for transesterification in acetonitrile. -Chymotrypsin which was co-lyophilized with hydroxypropylated - or -cyclodextrins retained more than 98% of its initial activity after 6 h incubation in acetonitrile.     

Cryptomonad biliproteins — an evolutionary perspective

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as 50000 kDa '₂ complexes which carry one bilin on the and three on the subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The subunit is encoded on the chloroplast genome, whereas the subunits are encoded by a small nuclear multigene family. The subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is primitive and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein and subunits.Abbreviations Chl chlorophyll - CER chloroplast endoplasmic reticulum - SSU rRNA small subunit ribosomal RNA     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.     

Anomeric preferences ofd-glucose uptake and utilization by cerebral cortex slices of rats

On aerobic incubation of rat cerebral cortex slices with anomers ofd-glucose and with 2-deoxy-d-glucose (2DG) for 5 min, the disappearance of -d-glucose from the incubation mixture was greater than that of -d-glucose and both anomers had a greater rate of disappearance than that of 2DG. In addition, there were significantly greater consumption of oxygen and production of lactate with the -anomer than with the -anomer. In similar experiments with³H-labeledd-glucose anomers and [1-³H]-3-O-methyl-d-glucose (3MG), the accumulation of [1-³H]--d-glucose (up to 5 min) by rat cerebral cortex slices was greater than that of [1-³H]--d-glucose. Although initially lower than that of the anomers, the accumulation of [1-³H]-3MG increased at a greater rate and, by 5 min of incubation, was greater than that of both glucose anomers. This preferential accumulation was seen to disappear when the slices were preincubated with 2DG (hexokinase inhibitor) or when the temperature of incubation was reduced to 20°C. Under those conditions the data with the glucose anomers were similar to those obtained with 3MG. Our data then suggested that the greater accumulation of -d-glucose than of -d-glucose by the slices was probably not due to differences in transport through brain cell membranes but rather to the preferential metabolism of the -d-glucose.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose