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PubMed2002

Enrichment of normal progenitors in counter-flow centrifugal elutriation (CCE) fractions of fresh chronic myeloid leukemia leukapheresis products.

OBJECTIVES: The aim of this study was to assess the suitability of a technique based on counter-flow centrifugal elutriation (CCE), which should allow one to enrich chronic myeloid leukemia (CML) patients' unstimulated native leukapheresis product (nLP) in CD34+ HLADR- cells and BCR-ABL negative cells. METHODS: Six newly diagnosed CML patients were subjected to leukapheresis, and the products were subfractionated with the use of CCE. nLP and all fractions were studied for the presence of CD34+ cells and a proportion of BCR-ABL fluorescence in situ hybridization (FISH)+ cells. RESULTS: CCE fractions with a high flow rate contained the highest proportion of CD34+ cells [mean (SEM) 6.89% (3.88)]. However, CD34+ cells present in low-rate CCE fractions showed a higher proportion of HLADR-[49.6% (13.5 in 70 mL min-1) and 21.5% (11.6 in 110 mL min-1)] than those in 170 mL min-1[3.2% (2.5)] and "rotor off" [3.4% (1.9)]. This was associated with lower proportions of BCR-ABL FISH+[8.1% (4.8) and 1.9 (1.7)] and smaller BCR-ABL to ABL transcript ratios [0.58 (17) and 0.26 (0.08) in 70 and 110 mL min-1] fractions as compared to 140 and 170 mL min-1 fractions [21.6% (5.2) and 31.6% (15.3) for BCR-ABL FISH+ cells and 0.75 (0.16) and 0.90 (0.24) for BCR-ABL/ABL]. Fractions with the lowest proportions of BCR-ABL-positive cells and the lowest BCR-ABL/ABL transcript ratios (110 mL min-1) contained from 1.3 x 106 to 82.7 x 106 (median: 3.97 x 106) CD34+ cells. CONCLUSIONS: In the present study we have shown that CCE may be used effectively to obtain nLP fractions enriched in normal hematopoietic progenitors.

Adult↗
PubMed1979

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation.

The S-phase-specific cytotoxicity of hydroxyurea (HU) was tested on synchronized murine fibrosarcoma (FSa) cells lodged in the lungs of C3Hf/Bu mice. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations. After elutriation, 8000 viable FSa cells from each fraction, along with 10(6) heavily irradiated tumour cells (unseparated) were injected i.v. into whole-body-irradiated mice (20 per group). Under these conditions, 95% of the injected cells, regardless of size or position in the cell cycle, are arrested in the lungs. Twenty minutes later, hydroxyurea (HU, 1 mg/g) was administered i.p. into 10 animals of each group. Fourteen days later the animals were killed, their lungs removed and fixed, and the number of macroscopic tumour nodules counted. Killing of the initially injected cells by HU, as evidenced by a reduction in lung colonies in treated animals, correlated with the precentage of S-phase cells in each fraction. The greatest effect, an 80% reduction in colony number, was seen in Fraction 8, containing the largest percentage of S-phase cells (65%). These results demonstrate the usefulness of this procedure as a rapid method for characterizing the phase specificity of chemotherapeutic drugs in vivo.

Animals↗
PubMed1981

Cell subpopulations dispersed from solid tumours and separated by centrifugal elutriation.

The degree of non-neoplastic host-cell infiltration was assessed in 3 in vivo-in vitro tumour models commonly used in radiobiological studies: EMT6/Ro mammary carcinoma, 9L/Ro tumour and KHT sarcoma. While the 2 former tumour models have been shown to be moderately to highly immunogenic when grown s.c., the KHT sarcoma is apparently non-immunogenic. Using differential staining on single-cell suspensions from enzymatically dissociated solid tumours, all 3 tumour types were found to contain large proportions (30-60%) of non-neoplastic host cells. The actual host-cell component found in the cell suspensions differed both in type and percentage for the 3 tumours studied. These host and neoplastic cells in the cell suspensions prepared from the solid tumours could be readily separated by centrifugal elutriation. After separation the clonogenic potential of the neoplastic cells was assessed, and was found to be higher than the clonogenic capacity of the unseparated cell suspension by a factor directly related to the host/neoplastic cell ratio. Even after the removal of the host cells, the clonogenic capacities of the neoplastic EMT6 and 9L tumour cells were lower than that of the corresponding in vitro sublines (approximately 30 vs 75%). However, in the KHT sarcoma the removal of the host cell component raised the plating efficiency to approximately 60%, which was similar to the value for the in vitro cell subline of this tumour.

Animals↗
PubMed1995

Rat brain glucose and energy metabolites: effect of +Gz (head-to-foot inertial load) exposure in a small animal centrifuge.

A unique small animal centrifuge with on-line physiological monitoring and brain tissue collection (in < 1 s) capability was used to investigate the effect of increasing +Gz levels, exposure duration, number of exposures, and time course of metabolic changes in the rat brain. To determine the +Gz tolerance, rats were exposed to +7.5 to 25 Gz (30 s each) and EEG was monitored. G-induced loss of consciousness (G-LOC) defined as isoelectric EEG (I-EEG) occurred only at +22.5 and 25 Gz within 14.5 +/- 3 s. To study the effect of increasing +Gz levels on metabolism, rats were exposed to either 0.5 (control) or +7.5 to 25 Gz (30 s each), and brains were collected 1 min postcentrifugation by freeze fixation. A significant increase in lactate (> or = +7.5 Gz) and a decrease in glucose, creatine phosphate (Cr-P), and ATP levels were observed at +15 Gz and higher. The effect of exposure duration was investigated by exposing the rats to +22.5 Gz for 15-60 s. Brain lactate levels increased six-fold while glucose decreased (75%) following the 60-s exposure. The level of Cr-P and ATP decreased significantly after the 15- and 30-s exposures with no further changes at longer +Gz exposures. For time course studies, brains were collected both during (5-35 s) and after (1-15 min) a +25 Gz exposure. A significant decrease in Cr-P occurred within 5 s, but changes in glucose, ATP, and lactate required 15 s. All metabolites returned to control levels within 3 min, except lactate and adenosine, which required 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Counterflow centrifugal elutriation as a method of T cell depletion may cause loss of immature CD34+ cells.

Counterflow centrifugal elutriation (CCE) is capable of separating cells on the basis of size. CCE has been used successfully to deplete allogeneic bone marrow (BM) grafts of T lymphocytes to decrease the risk of acute graft-versus-host disease. Previous studies have shown that more immature CD34+ cells in human BM tend to be smaller than more mature CD34+ cells. Human BM was subjected to CCE with the 4 ml standard chamber at constant rotor speed (2300 r.p.m.) and increasing flow-rate (14-23 ml/min, rotor-off). The eleven fractions collected were assayed for CD34+ and CD3+ cells, and for CFU-GM, HPP-CFC and long-term culture initiating cells (LTC-IC). The CD3+ T cells were enriched in the early (small-cell) fractions 14-17 ml/min. CD34+ cells were enriched in fractions 17-21 ml/min, and CFU-GM were concentrated in the same fractions. HPP-CFC and LTC-IC showed nearly identical CCE profiles, with enrichment in fractions 16-18 ml/min. When fraction < or = 17 ml/min was chosen as cut-off, the small-cell fraction contained 94.0% of all CD3+ cells, 44.4% of total cells, 33.2% of CD34+ cells and 34.7% of CFU-GM; however, 67.6% of HPP-CFC and 72.4% of LTC-IC were recovered in this small-cell fraction. These data suggest that T cell depletion through CCE as used by us, while losing only minor proportions of CD34+ cells and CFU-GM, carries the risk of losing the majority of more immature progenitor cells. This may lead to an increased risk of graft failure, in particular in HLA-mismatched transplants.

Antigens, CD34↗
PubMed1999

Counterflow centrifugation allows addition of appropriate numbers of T cells to allogeneic marrow and blood stem cell grafts to prevent severe GVHD without substantial loss of mature and immature progenitor cells.

Using counterflow centrifugation elutriation (CCE) lymphocytes can be separated from CD34+ populations based on size. Immature progenitors tend to be smaller than mature cells suggesting that CCE introduces loss of stem cells. We compared the separation of 12 PBSC with 16 BM transplants. Cells were separated in 12 fractions (3000-2200 r.p.m.) and the rotor off (RO) fraction. Separation patterns of BM and PBSC were comparable. B cells were collected in the high speed fractions followed by T and NK cells. In contrast, progenitor cells were collected in lower speed fractions. By adding successively T cell-depleted fractions to the RO fraction a BM transplant could be composed containing 0.7 x 10(6) T cells/kg and 90%, 89% and 68% recovery of CD34+, CFU-GM and BFU-E. PBSC were separated in four CCE runs inducing higher numbers of T cells in the graft (4.4 x 10(6)/kg) and 54% CD34+, 46% CFU-GM and 37% BFU-E recovery. Time of engraftment was not delayed and no graft failure was observed. The higher number of T cells was not associated with higher incidence of GVHD. Acute GVHD > or = grade III occurred in 0 of 16 BM and two of 12 PBSC recipients; extensive chronic GVHD was observed in four of 15 and three of nine recipients, respectively. To study immature cells in the graft, CD34 subpopulations and cells with long-term repopulating ability, determined using cobble-stone area formation (CAFC assay), were evaluated in each fraction. The separation patterns in BM and PBSC were comparable. Cells with mature and immature phenotype were enriched in lower speed fractions (mean recovery of 74% CD34+/CD13-/DR-). The CAFC week 2, 4 and 6 were also enriched in these fractions. These data show that the used CCE procedure is a reliable method to deplete T cells from stem cell transplants without substantial loss of immature and mature progenitors.

Adult↗
PubMed2006

Centrifugal extraction of plasma from whole blood on a rotating disk.

We present a centrifugal process for the extraction of plasma from sediment by a decanting structure, terminating with metered plasma which is readily available for subsequent on-disk processing. Our technique supplies 2 microl plasma from 5 microl of whole blood at moderate spinning frequencies of 40 Hz within 20 s, only. The residual cell concentration in the purified plasma amounts to less than 0.11%, independent of the frequency of rotation. A capillary duct connects the extracted plasma to subsequent on-disk processing units.

Blood↗
PubMed1968

The isolation of neurophysin-I and-II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient.

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11.56+/-1.63 and the pressor activity was 15.60+/-3.91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19.1 units of oxytocic and 21.1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.

Acid Phosphatase↗
PubMed1970

The isolation by isopycnic density-gradient centrifugation of two mitochondrial populations from livers of embryonic and fed and starved adult rats.

1. Mitochondria isolated from livers of fed adult, starved adult, and embryonic rats can be separated into three distinct bands by isopycnic density centrifugation on a sucrose density gradient. The least dense band (B1) has a mean buoyant density of 1.162 and consists mainly of disrupted mitochondria. The middle band (B2) has a mean buoyant density of 1.184. The most dense band (B3) has a mean buoyant density of 1.216. B2 and B3 consist of intact mitochondria. 2. The mitochondria in B2 and B3 have very similar protein/phospholipid ratios, virtually identical phospholipid and fatty acid compositions and similar specific activities for cytochrome oxidase, malate dehydrogenase and monoamine oxidase. Both fractions have very low glucose 6-phosphatase and acid phosphatase activities. 3. As isolated, adult rat liver mitochondria have electron-dense matrices (condensed forms); some embryonic liver mitochondria are condensed, but a significant proportion have dilated matrices. All B2 mitochondria are in the condensed form. B3 mitochondria from adult rats are condensed if fixed in their equilibrium-density sucrose, but when this is diluted rapidly to 0.25m they become swollen. Some B3 mitochondria from embryonic rats are condensed, the others have dilated matrices. They all swell if rapidly diluted to 0.25m-sucrose. B2 mitochondria retain their condensed form on dilution of the sucrose. 4. It is concluded that the matrix space of B2 mitochondria is almost totally inaccessible to sucrose, but that of B3 mitochondria is readily accessible to sucrose. 5. In liver from normally fed adult rats the B2 mitochondria predominate, whereas in starved rats B2 and B3 are present in approximately equal proportions. Mitochondrial preparations from embryonic liver consist predominantly of B3 mitochondria, but the proportion of these decreases progressively as development proceeds. 6. The B2 mitochondria from livers of fed adult rats can be converted into B3 mitochondria by incubation with 10mm-succinate and 10mm-phosphate. 7. Some B2 mitochondria are converted into B3 mitochondria by exposure to high concentrations of sucrose.

Animals↗
PubMed1971

The distribution of some hydrolases in glomeruli and tubular fragments prepared from rat kidney by zonal centrifugation.

1. A collagenase digest of rat kidney cortex was separated into four bands by zonal centrifugation. 2. Two of these bands were shown by light-microscopy to contain glomeruli and tubular fragments, which were free from each other and well separated from other renal material. 3. Protein, N-acetyl-beta-glucosaminidase, 5'-nucleotidase, l-leucine beta-naphthylamidase, leucine aminopeptidase, acid phosphatase and alkaline phosphatase were assayed across the gradient. 4. The greater proportion of these enzyme activities was recovered in the tubular fragments and acid phosphatase was the only enzyme detected in significant amounts in the glomeruli. 5. Tubular fragments and glomeruli were sedimented and multiple forms of beta-naphthylamidase, N-acetyl-beta-glucosaminidase, acid phosphatase and alkaline phosphatase were investigated by starch-gel electrophoresis.

Acid Phosphatase↗
PubMed1972

Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation.

The kinetics of incorporation of [(35)S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ;pulse-chase' radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ;pulse-chase' experiments over 5h did not demonstrate any precursor-product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ;pulse-chase' experiments there was again no evidence for precursor-product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

Animals↗
PubMed1972

Changes in nuclear protein during the cell cycle in cultured mast cells separated by zonal centrifugation.

1. Exponentially grown mouse mast cells (cell line P815, strain Y) were separated by zonal centrifugation on a Ficoll gradient. Fractions were allocated to different phases of the cell cycle according to the specific radioactivity of their DNA. 2. Histones were extracted and their thiol content was analysed. The proportion of reduced thiol increased in S phase, decreasing subsequently. 3. The phosphate content of histone F1 and of the other histones reached a peak in early and later S phase respectively. The incorporation of (32)P into these fractions showed a corresponding increase. 4. The timing of histone synthesis was examined. Incorporation of (14)C-labelled amino acids into the histone fractions took place at the same times as phosphorylation. 5. Acid nuclear proteins differ from the histones in incorporating labelled amino acids and (32)P fairly constantly through the cell cycle.

Amino Acids↗
PubMed1972

Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation.

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll-sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a(3) and cytochrome c+c(1) were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a(3) ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c(1)/cytochrome a+a(3) ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6-26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of alpha-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

Animals↗
PubMed1972

The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

Acid Phosphatase↗
PubMed1973

Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide.

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.

Acetamides↗
PubMed1973

The synthesis of ribonucleic acid in vivo in the nuclei of rat brain fractionated by zonal centrifugation.

1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [(3)H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [(14)C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.

Adenosine↗
PubMed1973

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation.

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).

Aging↗
PubMed1974

Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride.

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3x10(6)) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15x10(6)). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2x10(5) for mucoprotein A and 2.8x10(4) for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

Amino Acids↗