Antimutagenic Role of Autonomous 3′ → 5′-Exonucleases

Original and published data on the antimutagenic role of autonomous 3 5-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but are often involved in replicative complexes. AE overproduction in bacterial cells is accompanied by a sharp suppression of mutagenesis, whereas AE inactivation in bacteria and higher fungi results in the increase in mutation rates by two to three orders of magnitude. The combined action of AE and DNA polymerases substantially improves the fidelity of their functioning in vitro. The fidelity of nuclease-free DNA polymerases and increases by two to three orders of magnitude in the presence of AE. The fidelity of moderately processive DNA polymerase I increases by two orders of magnitude, and that of highly processive DNA polymerase increases by a factor of 5–10, although both these polymerases possess their own 3 5-exonucleolytic activity. In biochemical experiments, AE was shown to participate directly in the correction of errors made by DNA polymerase I. The presence of AE in multienzyme DNA polymerase complexes increases their fidelity by a factor of 5–10. A model of extrinsic proofreading by AE in DNA biosynthesis is proposed. An investigation of thirty objects from all three kingdoms of life (from archaea and bacteria to mammals, including humans) has shown that AE account for 30–90% of the total cellular 3 5-exonucleolytic activity. Therefore, AE increase significantly the intracellular ratio of 3 5-exonuclease to DNA polymerase activities in a wide phylogenetic variety of species, which always leads to the increasing fidelity of DNA biosynthesis.     

Relationships of Agropyron intermedium chromosomes determined by chromosome pairing and alcohol dehydrogenase isozymes in common wheat background

Summary The relationships of Agropyron intermedium chromosomes in two wheat-Agropyron addition series were determined. Chromosome pairing behaviour revealed that the alien chromosome in lines TAF-2 and L7 of Vilmorin-A. intermedium set are homologous to the alien chromosomes in lines P and C of the Caribo-A. intermedium set respectively. Localization of alcohol dehydrogenase isozyme genes in Vilmorin-Agropyron addition line L4 and in Caribo-Agropyron line O indicated relationships with wheat chromosomes of homoeologous group 4.     

Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport

Summary Intestinal lipid absorption and transport were investigated in albino rats. The observations point towards the existence of a continuity between plasma membrane invaginations and elements of the Golgi complex on its mature face. They also suggest a segregation of lipid droplets by paired Golgi membranes and plasma membrane invaginations. The following way for lipid transport is deduced: lipid droplets moving inside the smooth endoplasmic reticulum accumulate progressively and are condensed in Golgi cisternae of the forming face. Their limiting membrane ruptures and liberated lipid droplets are segregated by paired Golgi membranes of the mature face or by plasma membrane invaginations. Subsequently the inner of the two segregating membranes disappears while the lipid droplet is moved towards the intercellular space inside a canal communicating with this space. The suggestion is made that the Golgi apparatus is of double origin: one component representing a terminal plication of the endoplasmic reticulum; the second one—a terminal plication of the plasma membrane invagination. This concept explains the ultrastructural and histochemical differences between Golgi membranes of the forming and mature faces of the complex.     

Localization of individual subunits of protein kinase CK2 to the endoplasmic reticulum and to the Golgi apparatus

The protein kinase CK2 is composed of two catalytic - or - and two regulatory -subunits. In mammalian cells there is ample evidence for the presence of individual CK2 subunits beside the holoenzyme. By immunofluorescence studies using peptide antibodies which allow us to detect the CK2-, - and -subunits we found all three subunits to be co-localized with a 58 KDa Golgi protein which is specific for the Golgi complex. Subfractionation studies using dog pancreas cells revealed the presence of all three subunits of CK2 at the smooth endoplasmic reticulum (sER)/Golgi fraction whereas the rough endoplasmic reticulum (rER) harboured only the catalytic - and -subunits. We found that the microsomal preparation from dog pancreas cells contained CK2 which phosphorylated a CK2 specific synthetic peptide and which was heparin sensitive. Furthermore, we could immunoprecipitate the CK2-subunit that exhibited a kinase activity which phosphorylated a CK2 specific substrate and which was heparin sensitive. Protease digestion experiments revealed that the CK2 subunits were located on the cytosolic side of the rER and the sER/Golgi complex. Thus, we could demonstrate an asymmetric distribution of the CK2 subunits at the rER and sER/Golgi complex. Since the CK2- and -subunits exhibit a substrate specificity which is different from the CK2 holoenzyme one might speculate that the asymmetric distribution of the CK2 holoenzyme and the CK2 catalytic subunits may have regulatory functions.     

Hemoglobin vancouver [α2β2 73(E17) Asp→ Tyr]: Its structure and function

Summary Hemoglobin Vancouver is a new abnormal hemoglobin with an amino acid substitution of the normal aspartyl residue 73 of the chain by a tyrosyl residue. It was discovered in a man of Chinese descent in association with thalassemia. It was subsequently detected in a sister in association with normal Hb A. The oxygen affinity of the abnormal hemoglobin is decreased but its subunit interaction is normal. The Bohr effect may be slightly increased.This is the fourth abnormal hemoglobin to be found with a substitution at73. The others are Hb C-Harlem ( _{2 2} 6GluVal and 73 AspAsn), Hb Korle-Bu ( _{2 2} 73 AspAsn), and Hb Mobile ( _{2 2} 73 AspVal). Although Hb Mobile was found in the present studies to have a decreased affinity for oxygen, Hbs C-Harlem and Korle-Bu have been reported to be normal. These observations of functional differences for variants of73 added to earlier observations of the role of the normal73 residue to the aggregation of sickle deoxyhemoglobin indicate that this position of the molecule may be important in intra as well as intermolecular interactions.     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Die Steuerung der Schliessung und Öffnung des Plumula-Hakens bei Keimlingen vonLactuca sativa durch sichtbare Strahlung

Summary Dark grown seedlings ofLactuca sativa L. var. Grand Rapids (tip burn resistant strain) do not form an appreciable plumular hook. But a normal plumular hook, as is formed e. g. byPhaseolus vulgaris in complete darkness, can be induced by red light. It has been shown in this paper that the formation of the plumular hook in lettuce seedlings is controlled exclusively by the reversible red far-red reaction system (low energy reaction).The hook which has been closed by red radiation can be reopened by light. In this respect only blue and far-red radiation are effective. The hook can only be reopened if blue or far-red are applied with rather high irradiances over a relatively long time period (high energy reaction). The action spectrum and the kinetics of this high energy reaction show that this photoreaction is identical with the so called blue, far-red reaction system which we could demonstrate during recent years to be involved in the photomorphogenesis of the mustard seedling (Sinapis alba L.) and which apparently is also involved in many other photomorphogenic responses (cf.Hendricks et al. 1960). In mustard the low and the high energy reactions always function in a synergistic manner, in lettuce the opposite is true: The closing of the plumular hook is controlled by the reversible red, far-red system, the reopening of the hook is controlled by the blue, far-red system. In this way the two systems can be more clearly separated and characterized physiologically.

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Mit 12 Textabbildungen     

An inducible allantoinase and the specific toxicity of parabanic acid on allantoin utilization in aRhizobium species

ARhizobium sp. (strain NC 92) has been shown to be capable of utilizing uric acid, allantoin, allantoate, urea, and oxaluric acid as sole nitrogen sources. Allantoinase is repressible by NH ₄ ⁺ and inducible by allantoin and, less efficiently, by uric acid, oxaluric acid, and allophanate, but not by urea or parabanic acid. This allantoinase (purified 50-fold to homogeneity) is of 166 Kd M.W., is optimally active at pH 7.5, has a K_m of 4.16 mM and no requirement for sulfhydryl groups or metal ions, and is competitively inhibited by acetohydroxamate (K_i 9 mM). Parabanic acid is nontoxic toRhizobium NC 92 on inorganic N and is highly toxic to growth on allantoin N. Growth inhibition is reversed by supplemented allantoin, and suggestive evidence indicates that NC 92 metabolizes allantoin via the pathway: allantoin allantoate urea NH₃; allophanate is not an intermediate herein. Analysis of allantoinase induction indicates that the mandatory structural requirement is for a free urea moiety in an inducing molecule.     

Chromophore assignment in phycoerythrocyanin from Mastigocladus laminosus

The component spectra (maxima of absorption, circular and linear dichroism) of individual chromophores have been assigned for phycoerythrocyanin (PEC) trimer, monomer(s), and its subunits (-PEC and -PEC) by titration with p-chloromercury-benzene-sulfonate (PCMS), linear dichroism and photochemical transformations, as well as by deconvolution using a bilin line-shape spectrum based on the -84 phycoviolobilin-chromophore in the -subunit. The level ordering PVB--84 PCB--155 PCB--84 is the same irrespective of aggregation. Two different monomers () were observed. In 4 M urea, the spectra are appropriately weighted sums of the subunit spectra, whereas in the monomer obtained in 1 M KSCN, both -chromophores are red-shifted by 4–5 nm. Formation of trimer ()_{_3}gives considerable spectral changes: (1) the absorption is narrowed, which has been rationalized by excitonic coupling between neighbouring monomers, (2) the short wavelength part in the CD spectrum is missing and (3) a fourth band (+) at 528 in the LD spectrum appears. A deconvolution of the trimeric aggregation state using only the bilin line-shape model is not possible.     

The genetic structure of the incompatibility factors ofSchizophyllum commune: Comparative studies of primary mutations in theB factor

Summary Primary mutations in the three classes of theB factor were detected. The mutations map in the and loci of theB factor. The characteristic mating behavior of the mutant strains suggests functional differentiation of the and loci. Alternative interpretations are discussed.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

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Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

Enzymhistotopochemische Studien an inaktiven Salzdrüsen von Hausenten (Anas platyrhynchus)

Zusammenfassung In der sog. inaktiven Salzdrüse von Hausenten wurden die Glykosidasen -d-Glucuronidase, -d-Glucosaminidase, -Glucosidase und -d-Galaktosidase sowie die Hydrolasen unspezifische saure Phosphatase, unspezifische alkalische Phosphatase, Esterase, ATPase und Leucinaminopeptidase mit enzymhistochemischen Methoden untersucht. Die Drüsenzellen zeigen deutliche, wenn auch quantitativ unterschiedliche Esteraseaktivitäten. Besonders auffallend ist die hohe Aktivität der lysosomal lokalisierten sauren Phosphatase in den Epithelzellen der Zentralkanäle und Ausführungsgänge. Die Bedeutung der Befunde in bezug auf sekretorische und resorptive Leistungen der Salzdrüse wird diskutiert.

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Enzyme histochemical studies on the salt gland of ducks (Anas platyrhynchus)II. Cytochemical localization of some hydrolases

Summary Studying the site of hydrolytic enzymes in the salt gland of domestic ducks the glycosidases -d-glucuronidase, -d-glucosammidase, -glucosidase and -d-galactosidase as well as the hydrolases non-specific phosphatases, esterases, ATPase and leucinaminopeptidase have been investigated with histochemical techniques. The epithelia of the salt gland show distinct activities of non-specific esterases, that differ quantitatively. Furthermore we noticed a strong granular activity of non-specific acid phosphatase in the central canal and in the glandular duct system. The meaning of these findings in relation to secretion and absorption is discussed.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft (Ku-210/2).     

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.