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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03 :K2ab(L) : H2) an unusual amino sugar was isolated. By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the ammo sugar was characterized as a 2-amino- dideoxyaldose. The hydrochloride of the amino sugar, in comparison to those of 2-amino-2, 6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2, 6-dideoxy-D-mannose, 2-amino-2, 6-di- deoxy-D-allose, and 2-amino-2, 6-dideoxy-D-gulose, was studied with aid of ion exchange column chromatography. The N-acetylated amino sugar was compared with the N-acetylated reference amino sugars with regard to their relative mobihties in paper chromatography. The ammo sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer. On the basis of the results obtained the unknown amino sugar was identified as 2-amino- 2,6-dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L-configuration. This is the first report on the natural occurrence of L-rhamnosamine. [ABSTRACT FROM AUTHOR]

The role of Β-glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by the phosphoglucomutase for Β-glucose 1-phosphate (Β-phosphoglucomutase) has been examined in purified extracts of Euglena gracilis var. bacillaris. 1. The incubation of Β-glucose 1-[32P]phosphate with Β-phosphoglucomutase in presence of high concentrations (0.1 mM) of a commercial preparation of α-D(+)-glucose 1,6-bisphosphate (prepared synthetically) yielded a labelled compound running electrophoretically and chromatographically as the sugar bisphosphate. Specificity studies with Β-phosphoglucomutase and muscle phosphoglucomutase (α-phosphoglucomutase) strongly suggest that the compound formed is Β-glucose 1,6-[32P]bisphosphate. The results would also indicate that the commercial preparation of α-D(+)-glucose 1,6-bisphosphate contains Β-glucose 1,6-bisphosphate as contaminant. 2. When synthetic Β-glucose 1-phosphate preparations were purified by paper chromatography and then incubated with Β-phosphoglucomutase, no glucose 6-phosphate could be detected. On the other hand the reaction readily took place when challenged with the chromatographic fraction that runs as sugar bisphosphate. Upon analysis with etectrophoresis, chromatography and weak acid hydrolysis, and experiments with Β-phosphoglucomutase and α-phosphoglucomutase it was concluded that this compound is Β-glucose 1,6-bisphosphate. 3. It was established that the natural α-D(+)-glucose 1,6-bisphosphate not only fails to sustain the reaction catalyzed by Β-phosphoglucomutase but rather inhibits the reaction when added to the whole Β-phosphoglucomutase system, it was also demonstrated that Β-glucose 1,6-bisphosphate acts as inhibitor of the α-phosphoglucomutase system. 4. These results show for the first time that Β-glucose 1,6-bisphosphate is an active and necessary participant in the reaction catalyzed by Β-phosphoglucomutase. The role played by Β-glucose 1,6-bisphosphate in this reaction would be essentially the same as that of the α-anomer in the reaction catalyzed by α-phosphoglucomutase. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase flavocoenzyme (‘SD-flavin’), previously shown to be an 8α-substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 8α-Histidyl-riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH-fluorescence curve and behavior on thin-layer and paper chromatography, as well as paper electrophoresis. [This equation cannot be represented into ASCII Text] 3. Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)- and N(3)-imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6-N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α-[N(3)-histidyl]-riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin-imidazole bond and identification of the methyl-histidine liberated as 1-methyl-histidine. [ABSTRACT FROM AUTHOR]

4 UDP-trisaccharides present in the mammary gland and colostrum of the ewe were isolated and identified. The nucleotides, extracted by means of trichloracetic acid, were purified in the following way: 2 ion-exchange chromatographies on Dowex 1 (formate), filtration on Sephadex G-25, paper electrophoresis, pH 5, followed by paper chromatography in 3 different solvents. Using these fractionation techniques, 4 uridine derivatives (X1', X2', Y1', Y2') with positive reaction to the Warren neuraminic acid test, were obtained. UDP-5', which constitutes the nucleotidic part of the 4 derivatives, was defined more accurately after various acid or enzymatic analyses. The isolation of UDP-5' was made by ionexchange or paper chromatography and the total and labile phosphate was estimated. The Warren positive substances released from by neuraminidase or mild acid hydrolysis were identified as N-glycolyl-neuraminic acid for the X1', Y1' nucleotides, and N-acetyl-neuraminic acid for the X2', Y2' nucleotides. The behaviour of asialylated nucleotides to ion-exchange and paper chromatography is described as well as that of asialylated oligosaccharides. The 4 asialylated oligosaccharides contain galactose and an N-acetyl-D-hexosamine, the latter being N-acetyl-D-glucosamine in the case of X1' and X2', and N-acetyl-D-galactosamine in the case of Y1' and Y2'. Total analysis of UDPoligosaccharides shows a 1:1:1:1 ratio between uridine, N-acetyl-D-hexosamine, galactose, and neuaminic acid. The 1-P-trisaccharides and the trisaccharides were identified after phosphodiesterase hydrolysis followed by phosphatase hydrolysis. The N-acetyl-D-hexosamine carried the reducing group (sodium borohydride). Some structural characteristics of the 4 nucleotides were determined using β-galactosidase, neuraminidase, periodate, and periodate borohydride. Two of the nucleotides contain N-acetyl-glucosamine and are identical to those previously isolated from the goat colostrum. [ABSTRACT FROM AUTHOR]

Nicotinic acid in Rhodotorula glutinis is synthesized from tryptophan via N′-formyIkynurenine, kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid. Auxotrophic mutants, with blocked synthesis at various steps between tryptophan and 3-hydroxyanthranilic acid, can grow with 100 μg/ml anthranilic acid (concentration in the agar-agar). The usually required concentration of substances such as nicotinic acid or other substances after the blocked step is about 2 μg/mI. Auxotrophs with a block between 3-hydroxyanthranilic acid and nicotinic acid are not able to grow with anthranilic acid. Therefore mutants requiring nicotinic acid with a blocked step between tryptophan and 3-hydroxyanthranilic acid can synthesize nicotinic acid from anthranilic acid, a substance formed in the first step of tryptophan synthesis from chorismic acid, by eluding the blocked pathways The same ability has been found in prototroph revertants with the original block of the auxotrophs but with a second mutation in the gene of anthranilate synthetase. As a result of this mutation the feedback inhibition by tryptophan is disturbed and anthranilic acid is produced in excess. When the culture medium of the prototrophs is analysed by paper chromatography after adding anthranilic acid to the growing culture, 3-hydroxyanthranilic acid as one of the formed products can be found. Summarizing these facts it is evident, that there exists a "short-cut"-pathway of nicotinic acid synthesis in Rh. glutinis, which is realized by direct hydroxylation of anthranilic acid to 3-hydroxyanthranilic acid. This "short-cut"pathway however is only possible, when anthranilic acid in the cell is present in excess. [ABSTRACT FROM AUTHOR]

The purification and the amino acid sequence of the peptide containing the essential sulphydryl group of arginine kinase from Homarus vulgaris muscle is described. The fractionation of the tryptic digest of arginine kinase labelled with N-[1-14C]ethylmaleimide has been caused out using gel filtration, ion-exchange chromatography and paper chromatography. The composition of the radioactive peptide, isolated from the digest, is reported together with its amino acid sequence. A structural comparison is made between the peptide of arginine kinase and the corresponding active-cysteine containing peptide isolated from rabbit muscle creatine kinase. [ABSTRACT FROM AUTHOR]

Acidic capsular polysaccharide of Vibrio parahaemolyticus A55 (05:K15) was esterified with ethylene oxide and then reduced with sodium borohydride. Hydrolysis of the reduced material gave two hexosamines identical with mannosamine (2-amino-2-deoxy-mannose) and gulosamine (2-amino.2-deoxy-gulose) on paper and ion-exchange chromatograms. This fact suggested that the K15 antigen contained mannosaminuronic acid (2-amino-2-deoxy-mannuronic acid) and gulosaminuronic acid (2-amino-2-deoxy-guluronie acid). Direct acid hydrolysis of the antigen gave two 2-amino-2-deoxy-hexuronic acids. These two were identified as mannosaminuronic and gulosaminouronic acids by paper chromatography and paper electrophoresis, being compared with synthesized standard samples. Titration of acid form of the polysaccharide indicated that about 91.5% of the polysaccharide was composed of the N-acetyl-hexosaminuronic acids. [ABSTRACT FROM AUTHOR]

In 2000 ml of pooled human male plasma unconjugated 3β, 17β-dihydroxyandrost-5-ene as diacetate was identified. The identification was based on paper chromatography, acetylation followed by thin-layer chromatography and final mass spectrometry, Furthermore aliquot parts of the extract after paper-chromatography were subjected to a protein-binding assay to assure the 17β-hydroxyl configuration. [ABSTRACT FROM AUTHOR]

1. The purification of a halidohydrolase from cell-free extracts of Pseudomonas dehalogenans NCIB 9061 grown on glucose and monochloroacetate as carbon source, is described. 2. This enzyme catalyses the hydrolysis of some 2-halogen substituents on aliphatic carboxylic acids, releasing a halide ion and a hydrogen ion and causing inversion of the configuration. 3. The molecular weight as determined by chromatography on Sephadex G-100 was about 15000. 4. The optimum activity was at pH 9.4. Activity was rapidly and irreversibly lost below PH6.0. 5. The amino acid composition of the purified enzyme in its native state and after photooxidation in the presence of methylene blue is presented. 6. The variation of the kinetic parameters, V and Km between pH 7.0 and pH 10.0 suggest that a group with a pKa of about 7.8 is involved in its base form in the catalytic reaction. 7. The enzyme is rapidly inactivated by reaction with iodine and upon photooxidation in the presence of methylene blue, but the effect of thiol reagents suggests that a thiol group is not directly responsible for catalysis. 8. It is suggested that a histidine residue is implicated in the catalytic reaction and a mechanism is proposed which is consistent with the experimental results. [ABSTRACT FROM AUTHOR]

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudontoytas aeruginosa were isolated from the fraction containing the partially degraded polysaceharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed as necessary) by preparative high voltage paper electrophoresis The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaceharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate [ABSTRACT FROM AUTHOR]

Mild acid hydrolysis of the P+ lipopolysacharide of Salmonella minnesota mRz, a UDP glucose synthetase-less mutant, leads to the formatin of 5 degradation products whose structures were studied. Free 2-keto-3-deocyoctonate (KDO), KDO-7-phosphorylethanolamine, and fraction P- (Hep-Hep-KDO) have been identified previously as degradatino products of a P- lipoolysaccharide. Fractions P+ (Hep-(Hep-4-phosphate)-KDO) and PPN (Hep-(Hep-4-pyrophosphorylethanolamine)-KDO) occur only in the P+ mutant. It was shown that fraction P+ is not an artifact which was formed from fraction PPN during partial hydrolysis. The simultaneous occurrence of fraction P+ and PPN reflects microheterogenicity of the lipoolysaccharide. Possible reasons for this are discussed.

The presence of sulfated uronic acid residues in dermatan sulfate has been known for some time. In view of the copolymeric nature of pig skin dermatan sulfate, it was of interest to see whether the extra sulfate groups were attached to iduronic acid or glucuronic acid residues. Hyaluronidase-degraded dermatan sulfate was fractionated on Dowex-1 to yield fractions of varying sulfate content. It was observed that the degree of sulfation was inversely proportional to the glucuronic acid content. Oversulfated dermatan sulfate was subsequently treated with chondroitinase-AC to yield a completely glucuronic acid-free polymeric product still bearing all the extra sulfate groups. More direct evidence for the presence of sulfated iduronic acid moieties was obtained by Smith degradation of oversulfated, chondroitinase-AC treated dermatan sulfate. A fast-moving component isolated by preparative electrophoresis had the molar ratio sulfate: hexosamine 1.8:1 and contained exclusively L-iduronic acid. [ABSTRACT FROM AUTHOR]

This paper is a part of a project on the mechanism of activation of intestinal sucrase and of intestinal sugar transport by Na+. It reports a formal kinetic analysis of the following aspects: 1. Investigation on the apparent Km for sucrose hydrolysis in intestinal rings and homogenates does not indicate the existence of any gross diffusion barrier for sucrose between lumen and brush-border bound sucrose, Therefore, it is possible to compare kinetic parameters of solubilized sucrase with kinetics parameters of the intestinal sugar uptake system. 2. Intestinal sucrases from hamster, rat, Greek turtle, man and rabbit (the latter two reported in previous papers) are all activated by Na+. The modes of activation and the apparent Michaelis constants for Na+ are different for each species considered.3. K+ and Li+ have effects similar to those of Na+. 4. The Ksapp = f (Na+]) functions indicate a non-compulsory reaction sequence in Na+-activation of sucrase. 5. Hamster and rat sucrases show a cooperative interaction between homologous sites. Papain solubilization does not make it disappear. 6. The pH-activity curves are different in each species. In some species the percent activation by Na+ depends on the pH. 7. The Kochsaft of small intestine contains low molecular weight substance(s) which decrease the color yield in the glucose-oxidase/peroxidase assay for glucose. [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [ABSTRACT FROM AUTHOR]

From the venom glands of honey bees fed with different radioactive amino acids, labelled promelittin could be isolated. Extensive analysis of this precursor peptide has led to the conclusion that it contains the entire sequence of melittin, including the C-terminal glutamic acid diamide. At the amino end, promelittin was found to be heterogeneous. As compared to melittin, the main species contains eight additional amino acids. Other species of different chain length are present in varying amounts. The structure of the main component can be formulated as Glu-Pro-Glu-Pro-Asp-Pro-Glu-Ala-melittin(1–26). The methodology used for determining the sequence of radioactive peptides is discussed. [ABSTRACT FROM AUTHOR]

A hitherto unknown amino sugar was discovered in lipopolysaccharides isolated from strains of chemotype III of the photosynthetic bacterium Rhodopseudomonas palustris. The sugar (2-3% of the lipopolysaccharides dry weight) is detected as a shoulder on the descending part of the glucosamine peak obtained when acid hydrolysates of the lipopolysaccharides are passed through an amino acid analyzer. It was separated from the other amino sugars of the lipopolysaccharide (i,e. glucosamine, galactosamine, quinovosamine) by high voltage paper electrophoresis and subsequent paper chromatography. The mass spectrometric analysis of the isolated sugar as deuterium-reduced alditol acetate revealed the presence of a 6-0-methyl-2-amino-2-deoxyhexose. Demethylation of the isolated sugar afforded glucosamine and the demethylation of its ninhydrin degradation product yielded arabinose, which showed that the parental amino sugar is glucosamine. Authentic 6-O-methyl-D-glucosamine exhibited identical properties in paper chromatography and high-voltage electrophoresis as did its alditol acetate derivative in gas-liquid chromatography and mass spectrometry. These data prove that the unknown amino sugar in lipopolysaccharides of Rhodopseudomonas palustris is 6-0-methyl-glucosamine. The strong positive optical rotation of the isolated sugar indicates its D-configuration. 6-0-Methyl-D-glucosamine is part of the O-specific chain and not of the lipid A moiety of the lipopolysaccharide. Periodate-oxidation studies revealed that the sugar, like the other amino sugars of this lipopolysaccharide, does not occupy non-reducing terminal positions to any appreciable extent. [ABSTRACT FROM AUTHOR]

Evidence is presented to show that an alkaline hydrolysis product from 40-S and 28-S RNA of Xenopus laevis is a tetranucleotide containing three adjacent 2'-O-methylations. This is the longest such sequence yet reported and is believed to be Gm-Am-Am-Ap. [ABSTRACT FROM AUTHOR]

In the previous paper in this series it was shown that a pure flavin pentapeptide isolated from hepatic monoamine oxidase contains I mole each of serine and tyrosine and 2 moles of glycine, as well as an additional amino acid which is covalently linked to the 8α carbon of riboflavin. This amino acid has been identified as cysteine and the linkage to the flavin as a thioether on the basis of positive chloroplatinic and negative iodine-azide tests, performic acid oxidation or reduction with zinc, followed by acid hydrolysis, which yield cysteic acid or cysteine, respectively. The fluorescence properties of the flavin from monoamine oxidase agree with those expected for a thioether and its oxidation products. In regard to optical and electron spin resonance spectra, chemical stability, and fluorescence characteristics the flavin peptide isolated from the enzyme agrees excellently with the properties of synthetic cysteinyl 8α-riboflavin. [ABSTRACT FROM AUTHOR]

The fate of the hydrogen atoms at C-3 and C-4 of uridine diphospho-D-glucuronic acid (UDPGlcUA) in the course of its conversion to uridine diphospho-apiose (UDP-api) was investigated. The specifically labeled UDP-GlcUA substrates were prepared enzymatically from D-[3-³H]glucose and D-[4-³H]glucose by the combined action of hexokinase, phosphoglucomutase, UDPGpyrophosphorylase, and UDPG dehydrogenase. UDP-[U-14C, 3-³H]GlcUA, UDP-[U14C, 4-³H]GlcUA, and UDP-[U14C, 3,4-³H2]GlcUA were separately converted to UDP-Api by an enzyme preparation from Lemna minor. In order to obtain an apiose (3-C-hydroxymethyl-D-erythrofuranose) derivative which was reasonably stable and which could be produced without passing through the aldehydo form, the apiosyl radical of UDP-Api was enzymatically transferred to 7-glucosylapigenin by an enzyme preparation from parsley. The product of this reaction, apiin (7-O-[β-D-apiofuranosyl-(1→2)-β-D-glucosyl-5,7,4'-trihydroxyflavone), was subjected to mild periodate oxidation to convert the branch hydroxymethyl group of the apiosyl residue to formaldehyde. Approximately 14-16% of each of the substrates was converted to UDP-Api. The transfer of the apiosyl moiety to 7-glucosylapigenin was essentially quantitative in all cases. The isolated apiin was shown to have retained 53.5% of the tritium from the substrate labeled at C-3, 99% from the substrate labeled at C-4, and 73% from the substrate labeled at C-3,4. All of the tritium and one-fifth of the 14C of apiin produced from the variously labeled substrate was accounted for in the formaldehyde-dimedone derivative. These results establish the occurrence of a hydride shift from C-4 of UDP-GlcUA to C-3' of apiose in the biosynthesis of UDP-Api from UDPGlcUA. [ABSTRACT FROM AUTHOR]

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylie acid residues to deoxyoligonucleotides. This reaction allows the specific labeling of deoxyoligonucleotides at the 3′-ends if [α-32P]ATP is used as substrate. In order to eliminate the product of the addition of two riboadenylie acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl [32P]pAr is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide-3′-[32P]monophosphates corresponding to the 3′-ends of the deoxyoligonucleotides. The riboadenosyl residues at the 3′-termini of the monoaddition products also can be eliminated by treatment with periodate and cyclohexylamine whereupon deoxyoligonucleotides specifically labeled at the 3′-termini with [32P]phosphomonoester groups are obtained. The sensitivity of the method and the faithfulness in the identification of the 3′-terminal deoxynucleoside residues could be tested using as little as 0.1 A250 units (5 μg) of oligodeoxynucleotides of specific base sequences. [ABSTRACT FROM AUTHOR]

An investigation on the sugar compositions of lipopolysaccharides (O antigens) derived from 45 well—defined Citrobacter serotypes was carried out. As a result, a chemical classification into 20 chemotypes was achieved, of which 11 were identical with chemotypes occurring in Salmonella and Escherichia coli. All Citrobacter lipopolysaccharides contained glucosamine, glucose, heptose, 2-keto-3-deoxyoctonate and galactose, the latter, in some cases, in very small amounts. These sugars probably represent building blocks of the basal core polysaccharides, whose structures however may not be identical for all Citrobacter lipopolysaccharides. In addition to the basal sugars, the following monosaccharides were detected as constituents of the various Citrobacter lipopolysaccharides; D-xylose, D-mannose, fucose, rhamnose, 6-deoxylatose, 4-deoxy-D-idose, abequose, galactosamine, D-fucosamine, 3-amino-3,6-dideoxy-glucose and -galactose. The sugar compositions of serologically related Citrobacter and Salmonella O antigens were compared. While some cross-reacting pairs belonged to the same chemotype, other proved to be distinct with respect to their sugar constituents. [ABSTRACT FROM AUTHOR]

Mild acid hydrolysis of the glycolipid derived @om a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and L-glycero-α-D-mannoheptosyl-l,5-KDO. [ABSTRACT FROM AUTHOR]

If no NADPH is available for reduction, fatty acid biosynthesis is blocked at the stage of the acetoacetyl-acyl carrier protein intermediate. In this case highly purified fatty acid synthetase from baker's yeast catalyzes, in a derailment reaction, the formation of triacetic acid lactone from acetyl- and malonyl-CoA. The identity, of the product was shown by the enzymatic incorporation of radioactivity from [1-14acetyl-CoA and from recrystallization to constant. specific radioactivity. In paper chromatography, thin layer chromatography, and ionophoresis both the chemically synthesized triacetic acid lactone and the enzymatically formed compound migrated with the same RF-values and electrophoretic mobility. By an oxidation with chromic acid according to the procedure of Kuhn-Roth it could be demonstrated that the radioactivity of [1-14C]acetyl-CoA is incorporated only into the C-6 position of triacetic acid lactone. Free triaceti acid and tetraacetic acid lactone do not seem to be formed by fatty acid synthetase. The ratio of the malonyl-CoA utilization for the synthesis of palmitoyl- and stearoyl-CoA to that for the synthesis of triacetic acid lactone was found to be about 90:1. The significance, of these findings is discussed. A chemical mechanism for the formation of triacetic acid lactone by fatty acid. synthetase is proposed. [ABSTRACT FROM AUTHOR]

Large oligonucleotide fragments obtained by partial digestion of 32P labelled E. coli tRNAF with pancreatic and T1 ribonuclease have been separated and characterized. The information provided by these analyses has permitted the derivation of the total sequence of tRNAF. [ABSTRACT FROM AUTHOR]

Tryptic digestion of peptide A, the C-terminal fragment of thioredoxin, obtained by cyanogen bromide cleavage, resulted in the isolation of nine peptides. The complete amino acid sequence of each was determined, and the N-terminal and C-terminal fragments of peptide A were identified. Together the nine tryptic peptides accounted for all 71 residues of peptide A. Hydrazinolysis and studies with carboxypeptidase A established -Asn-Leu-Ala to be the C-terminal sequence of peptide A and of thioredoxin. [ABSTRACT FROM AUTHOR]

Tryptic and chymotryptic digestion of peptide B, the N-terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: -Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys-Met-. This sequence supports earlier conclusions dragon from fluorescence studies of thioredoxin, which indica ted a close relationship between one or both tryptophan residues and the disulfide bridge of the active site. Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C-terminal fragment (peptide A). [ABSTRACT FROM AUTHOR]

Pure mucin isolated from canine submaxillary glands was treated with alkaline borohydride and products of degradation were fractionated by a procedure that included ion-exchange chromatography, gel filtration, high-voltage electrophoresis and paper chromatography. Seven acidic, reduced oligosaccharides were isolated being divided into two distinct types: one, containing sialic acid and no glucosamine or sulfate (type A) and the other containing glucosamine and sulfate but no sialic acid (type B). The most complete oligosaccharide of type A designated component (I) was a tetrasaccharide α-L-fucopyranosyl-(1 → 2)-D-galactopyranosyl-[N-acetylneuraminyl-(2 → 6)]-2-acetamido-2-deoxy-D-galactitol. The most complex oligosaccharide of type B designated component (V) has a hexasaccharide with the following structure: α-L-fucopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3, 4, or 6)-β-2-acetamido-2-deoxy-D-glucopyranosyl 3 or 4 sulfate-(1 → 6)-[α-L-fucopyranosyl-(1 → 2)]-β-D-galactopyranosyl-(1 → 3) 2-acetamido-2-deoxy-D-galactitol. All the other acidic isolated oligosaccharides were derived from these two main components. No oligosaccharide containing both sialic acid and glucosamine sulfate could be demonstrated. Based on these structural features, a possible biosynthetic pathway leading to the two different oligosaccharide chains is discussed. Variations in ratios of sugars observed in canine submaxillary mucin and especially the reciprocal relationship between sialic acid and fucose noted earlier (1962) by Dische. Pallavicini, Kavasaki, Smirnow, Cizek and Chien may reffect variations in the proportions of these two types of oligosaccharides. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

The isolation and amino-acid and carbohydrate composition of three tryptic glycopeptides, containing 16, 17 and 20 amino-acid residues, respectively, from the neuraminidase-treated major form of human ceruloplasmin have been described earlier. It has also been suggested that the peptides possibly represent all of the carbohydrate-binding sites in the protein. The present paper describes the amino-acid sequences of these peptides as obtained by stepwise Edman degradation and carboxypeptidase digestions of the whole peptides and their fragments after cleavage by chymotrypsin, pepsin and thermolysin. The results confirm that the carbohydrate in all cases is attached via an asparagine (or aspartic acid) residue, that is followed by an X-Thr/Ser sequence. No sequence homologies are found when the peptides are compared to each other or to other glycopeptides of known sequence. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharides of Vibrio cholerae strains Inaba and Ogawa, an unusual amino sugar was isolated. By sequential oxidation with sodium metaperiodate and hypoiodide and by oxidation with periodate followed by detection of the acetaldehyde formed, the amino sugar was characterized as a 2-amino-dideoxyaldose. The hydroehloride of the amino sugar was compared with those of 2-amino-2,6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2,6-dideoxy-D-mannose, 2-amino-2,6.di-deoxy-D-gulose, and 2-amino-2,6-dideoxy-D-allose in an amino acid analyzer. The amino sugar alcohols were studied by paper electrophoresis in molybdats buffer. From the results obtained the amino sugar was identified as 2.amino-2,6-dideoxyglucose (quinovosamine). Its optical rotation indicated it to be of the D-configuration. [ABSTRACT FROM AUTHOR]

With the aim of establishing a starting point for the determination of the complete amino acid sequence of the major parvalbumin from hake muscle, the peptides resulting bom the tryptic digestion of the S-sulfo-derivative and of the performic, acid-oxidized protein were examined. A total of 20 peptides were thus obtained m sufficient quantity and m a state of purity adequate for further studies. Their complete or partial sequence is reported. When due account is taken of obvious overlaps, the total of these peptides fully accounts for the composition of the whole molecule with the exception of one alanine residue, and for the number of peptides expected from the presence of 12 lysines and 1 arginine in this parvalbumin. [ABSTRACT FROM AUTHOR]

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (08:K27-:H-) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 450% aqueous phenol at 65 °C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2-keto-3-deoxy-mannosulonic acid (0.8%). Polysaccharide II was obtained in 0.8–1% yield (based on dry bacteria). It contained 99.2% mannose. With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43 °C for polysaccharide I and 42 °C for polysaccharide II. Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas-liquid chromatography and mass spectrometry indicated that in both polysaccharides two-thirds of the mannose units were substituted at C-2 and one-third at C-3. These results were confirmed by periodate oxidation. From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry. It is concluded that the poIysaccharides I and II contain about 20 repeating units of α-mannosyl-1,2-α-mannosyl-1,2-α-mannose which are joined through &alph;-1,3 linkages. [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Polynucleotide phosphorylase from Escherichia coli cells catalyzes the transfer of a single nucleotidyl residue from each of the 2'(3')-O-isovalerylesters of ADP, GDP, CDP and UDP to the initiator oligonucleotide, A-A-A. The products of these reactions are identified as the 2'-(3')-O-isovaleryl esters of the corresponding tetranucleotides A-A-A-A, A-A-A-G, A-A-A-C and A-A-A-U. The 2'-(3')-O-isovaleryl residue can be removed from the product by treatment with aqueous methanolic ammonic under conditions that do not significantly damage the oligonucleotide chains. The monoaddition of 2'(3')-O-acyl esters of nucleoside diphosphates to an oligonucleotide acceptor is proposed as a method for the stephwise synthesis of oligoribonucleotides of defined sequence. [ABSTRACT FROM AUTHOR]

When the lipopolysaccharides of two Salmonella R mutants were hydrolyzed in 0.5 N HCl at 37° for 7 to 24 h, a previously undescribed sugar was liberated. This sugar has been identified as 4-amino-4-deoxy-L-arabinose by comparison of the free amino sugar, its two reduced derivatives, its two N-acetylated derivatives, and its methyl glycoside with the authentic amino sugar and its corresponding derivatives on thin layer, gas liquid, and column chromatography. A survey of about 50 enterobacterial lipopolysaccharides revealed that this amino sugar is present in a large percentage of the preparations. The preparation of 4-amino-4-deoxyarabinose by the reduction and limited periodate oxidation of 2-amino-2-deoxymannose is described. [ABSTRACT FROM AUTHOR]

Porcine secretin is a heptacosapeptide with the amino acid sequence His-Ser-Asp-Gly-Thr- Phe -Thr- Ser -Glu -Leu -Ser -Arg -Leu -Arg -Asp-Ser -Ala -Arg -Leu -Gin -Arg -Leu -Leu-Gln-Gly-Leu- Va1NH2. Fourteen of its twenty-seven amino acids occur in the same position as they do in porcine glucagon. [ABSTRACT FROM AUTHOR]

Extracts of the hypobranchial body of two prosobranchiate molluses of the Muricidae family were examined for their content of choline esters, as well as of precursors and metabolites of these esters. The hypobranchial gland of Concholepas concholepas contained, in addition to large amounts of urocanylcholine (murexine) and senecioylcholine, appreciable amounts of free choline, free urocanic acid and ethyl urocanate. All these compounds, and especially the choline esters, were predominantly located in the dye-secreting area of the hypobranchial gland. As much as 3% of the wet weight of the purple area was accounted for by murexine and seneciocylcholine. The only choline ester found in the hypobranchial body of Thais cholate was senecioylcholiine; derivatives of urocanic acid were lacking. The occurrence of ethyl urocanate in the hypobranchial gland of Concholepas concholepas demonstrates that not only choline but also other alcohols may be esterified by urocanic acid. [ABSTRACT FROM AUTHOR]

The endotoxically active cell wall glycolipid of a highly deficient heptose-less R mutant of Salmonella minnesota, consisting of 2-keto-3-deoxyoctonic acid (KDO) and lipid A, was subjected to hydrazinolysis. Besides fatty acid hydrazides, a water-soluble main fraction was obtained, containing KDO, glucosamine, and ester phosphate and free phosphorylethanolamine. The application of two procedures of partial hydrolysis, followed by paper electrophoretic separation, led to seven oligosaccharides. Their structures were evaluated with the aid of the following analytical methods: further acid degradation, borohydride reduction, ninhydrin degradation, the Morgan-Elson reaction, treatment with phosphomonoesterase and N-acetylglucosaminidases. The results show that the backbone of lipid A contains phosphorylated glucosaminyl→glucosamine disaccacharide units, probably linked β-1 ,6, to which a trisacoharide composed of three KDO residues is linked ketosidically to form a pentasaccharide which appears to be a common structure in many Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

For the monosialo-lactoisohexaosyl-ceramide, a ganglioside from human spleen, the following structure was established: NeuNAcα2 → 3Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4Glc → Cer. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharide of the gram-negative bacterium Rhodopseudomonas capsulata (Athiorhodaceae) a lipophilic sugar was isolated by chromatographic procedures and characterized by mass spectrometry as belonging to the group of 3-O-methyl-6-deoxy-hexoses. Demethylation showed that the parental sugar had the same properties in borate electrophoresis as rhamnose. Comparison with an authentic sample of 3-O-methyl-rhamnose in gas liquid chromatography, paper chromatography and borate electrophoresis showed that the sugar under examination is 3-O-methylrhamnose (acofriose). Investigation of its optical rotation indicates that the sugar has the L-configuration. L-Acofriose (3-O-methyl-L-rhamnose) has not hitherto been found in lipopolysaccharides of gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr. respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Osidatlon at 40°C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0°C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand. has a positive band at 340 nm, as m FAD. but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein. [ABSTRACT FROM AUTHOR]

Semliki-Forest-virus 42-S virion RNA is translated in a mouse ascites cell-free system. One of the major polypeptides made in vitro has been identified as Semliki-Forest-virus capsid protein by co-electrophoresis on dodecylsulfate -- polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell-free system has been characterised as envelope protein by peptide analysis but it does not co-electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non-glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell-free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell-free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki-Forest-virus 42-S RNA are discussed. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [ABSTRACT FROM AUTHOR]

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4 °C in 0.017 M K2HPO4 or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [14C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [14C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane-bound transpeptidase. The same rate constant (about I × 10-4s-1) for the dissociation of the benzylpenicillin membrane complex at 37 °C and in 0.017 M K2HPO4, was calculated either from the release of the radioactivity (using [14C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high-affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37 °C and in 0.017 M K2HPO4 at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10-3 to 0.73 ×10-4 s-1. The radioactivity released through the dissociation of [14C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [14C]-benzylpenicilloic acid both by paper electrophoresis and thin-layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule. [ABSTRACT FROM AUTHOR]

I. This paper describes some features of the protein synthesis in isolated rat liver mitochondria and reports on the stability of the product of mitochondrial protein synthesis in vitro. 2. Chase experiments performed after a 30-min pulse of [14C]leucine indicated that a large part of the product is degraded to an acid-soluble form (labile product) almost as rapidly as it is terminated and released from the mitoribosome. Only a small proportion of the completed product (between 20 and 40 %)is conversed in acid-insoluble form (stable product). 2. Both labile and stable products have an exceptional degree of hydrophobicity, shown by their marked resistance to acid and alkaline hydrolysis and by their solubility in organic solvents. 4. A 14C-labelled peptide with an apparent molecular weight of 12000-13000, as assessed by gel electrophoresis in the presence of dodecylsulfate, is released after heat treatment of mitoribosomes isolated from mitochondria pulsed with [14C]leucine. [ABSTRACT FROM AUTHOR]

1. Adenylate kinase (ATP: AMP phosphotransferase) has been purified 490-fold from porcine muscle with a final yield of 60 mg/kg muscle. 2. The amino-acid composition is Asp11 Asn2, Thr14, Ser11, Glu19,, Gln6, Pro6, Gly19>, Ala6, Cyst2, Val17, Met6, Ile9, Leu18, Tyr7, Phe5, Lys21, His2, Arrg11. 3. The protein molecules a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine residue at the C-terminus. 4. Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α-protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained. 5. The primary structure of porcine adenylate kinase was given. [ABSTRACT FROM AUTHOR]

The incorporation of radioactivity in vitro from UDP-[14C]glucose into endogenous acceptors in a crude enzymatic preparation from Acetobacter xylinum is described. A fraction of the total incorporated radioactivity could be extracted with organic solvents, like butanol. The remainder was mostly solubilized by hot alkali, leaving an insoluble fraction. No incorporation was observed in the absence of magnesium ions. Mn2+ could only partially replace Mg2+, and had an inhibitory effect when both ions were present. Analysis of the butanol-soluble fraction permitted the isolation of at least three components characterized as: lipid-diphosphate α-glucose, lipid-diphosphate α-cellobiose and lipid-monophosphate β-galactose. In the presence of [β-32P]UDP-glucose the system incorporated radioactivity only into the diphosphate derivatives, indicating the transfer of a α-glucose 1-phosphate group from UDPglucose. Experiments with doubly labelled substrate confirmed these results. The role these compounds might play in polysaccharide biosynthesis is discussed. [ABSTRACT FROM AUTHOR]

The maximal potential activity of UDP-glucuronyltransferase is constrained by the structure of the phospholipid environment in intact microsomal membranes. This constraint can be relieved by treatment of microsomes with phospholipase A. As shown by the data in this paper, however, relief of constraint is associated with a loss of specificity in the binding of UDP-sugars at the UDP-glucuronic acid site of UDP-glucuronyltransferase. As a result, several UDP-sugars which have no effect on the activity of the untreated enzyme act as inhibitors of the unconstrained, phospholipase A-treated form of UDP-glucuronyltransferase. In addition to the loss of specificity of substrate binding, the phospholipase-A-treated form of UDP-glucuronyltransfease cannot be activated by UDP-N-acetylglucosamine, which is a positive K-type of allosteric effector for the untreated form of the enzyme. UDP-N-acetylglucosamine, in fact, is an inhibitor of the phospholipase-A-treated form of the enzyme. In contrast to the effects of other UDP-sugars, inhibition by UDP-N-acetylglucosamine seems to result from the binding of UDP-N-acetylglucosamine at an allosteric site rather than the active site. Inhibition of the phospholipase-A-treated form of UDP-glucuronyltransferase by UDP-N-acetylglucosamine and other UDP-sugars is additive. [ABSTRACT FROM AUTHOR]