Sephacryl S-200 Gel Research Articles

Molecular Composition of Starches from Growing Potato Tubers

AbstractThe changes in the molecular distribution of potato starch during the growth of tubers is determined by GPC (Sephacryl gel systems). After n‐butanol fractionation of amylose and amylopectin an efficient statement of the molecular weight alteration of the starch components is observed. Amylopectin of young immature tubers shows a narrow highmolecular weight distribution whereas the amylopectin of mature tubers have a broader distribution with low‐ and highmolecular weight portions. Also the branched structure is different regarding starch of small and big tubers. Amylose shows a continous increase of the molecular weight. The range up to 0.2 · 106 Dalton is diminished, the molecular weight range over 1.2 · 106 Dalton is increased. The range in between rests nearly constant. These changes in the molecular composition are observed by different potato varieties, Conny and Sieglinde. Clear differences between the amyloses and amylopectins of these two varieties are noticed. The starch components of Sieglinde have always lower molecular weights as Conny starches, just so the overlapping range is much broader.

Incorporation of the purified human placental insulin receptor into phospholipid vesicles.

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.

Neutrophil granulocyte functions in severely burned patients

Burns wound sepsis is not only the most common but also the most severe complication following extensive thermal injury. One conceivable explanation of this problem is a reduced capacity of the polymorphonuclear neutrophil leucocytes of these patients to combat the invading microbes. Fifty patients (42 male and 8 female) with deep dermal burns, covering 20–90 per cent of the total body surface area, were investigated from immediately after the injury until death or until healing of the wounds. The following functions of the neutrophil granulocytes were studied: (1) chemotaxis and random migration utilizing a modified Boyden chamber technique, (2) phagocytosis of Staph. aureus and IgG-coated latex particles, (3) bactericidal capacity, e.g. killing of Staph. aureus and (4) the neutrophil granulocyte content of: myeloperoxidase, lactoferrin, and chymotrypsin-like cationic protein. The presence of stimulators and inhibitors of the granulocyte functions was studied using gel filtration of the patient's serum on Sephacryl gel columns. Sera from all patients obtained within the first 1–3 days post-burn contained significantly increased amounts of heat-labile chemokinetic stimulating activity. Sera obtained between days 4 and 10 after injury contained significantly decreased amounts of heat-stable chemokinetic stimulating activity. Reduced chemokinetic activity was found during the third and fourth weeks following major burns (⩾40 per cent) due to the presence of one or both heat-stable chemokinetic inhibitory activities. During the second week post-burn patients with burns larger than 40 per cent of the body surface area who showed an inhibition of chemotaxis, also had defects in phagocytosis, and often impaired bactericidal capacity concomitant with lower contents than normal of the granular enzymes. A hyaluronic acid preparation in low concentrations was found to counteract the migration inhibitory effect demonstrated in vitro in sera from patients with severe burns. Based upon these results a series of patients with severe burns and impaired functions of the neutrophil granulocytes have been treated with small amounts of this hyaluronic acid preparation subcutaneously. Very promising results have been noticed, similar to those found in vitro.

Monoclonal antibodies to human plasma protein X alias complement S-protein.

Protein X alias complement S-protein was isolated by dissociation from purified XC5b-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immunoprecipitate of alpha 2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of alpha 1-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monoclonal anti-Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500-700 micrograms/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.

Malic enzymes of salmon trout heart mitochondria: separation and some physicochemical properties of NAD-preferring and NADP-specific enzymes

Mitochondria isolated from the heart of the Baltic salmon trout Salmo trutta contain two distinct malic enzymes. One of these enzymes (NAD-preferring malic enzyme) catalyses the oxidative decarboxylation of malate in the presence of either NAD or NADP. The specific activity with NAD was six times that with NADP as coenzyme. The second enzyme is specific for NADP. These two malic enzymes have been separated by: ion exchange chromatography of DEAE-Sephacel, affinity chromatography on 2',5'ADP-Sepharose 4B, gel filtration on Sephacryl S-300 and polyacrylamide gel electrophoresis. The mol. wts of the two native malic enzymes determined by gel filtration were found to be 280,000 and 190,000 for NAD-preferring and NADP-specific malic enzyme, respectively. Chromatofocusing revealed the isoelectric points of the two enzymes at pH 5.45 and 5.85 for NAD-preferring and NADP-specific malic enzyme, respectively.

Molecular weight measurement of human satietin

Attempts were made to estimate the actual molecular weight of satietin, an endogenous food intake inhibitor, by means of classical and high-performance gel filtration and gel electrophoresis. Satietin, which proved to be a glycoprotein, was isolated from human plasma. Sephacryl S-300 gel and TSK-GEL G 3000 SW columns were used as media for gel filtration and gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), respectivley. A relatively good correlation was established between the molecular weight measurements carried out by different methods, although satietin as a carbohydrate-rich glycoprotein gave an underestimated value by gel filtration when the calibration graphs were constructed with simple proteins. The molecular weight of satietin was determined to be about 43,000 daltons by SDS-PAGE and 29,000 daltons by gel filtration when using a buffer containing 1% of sodium dodecyl sulphate.

Lectin-like factor and co-factor in serum from cystic fibrosis heterozygotes

Previous attempts to use an assay for a serum lectin-like factor as a carrier test for the cystic fibrosis gene have not been successful because food Intake and effects of serum storage have interfered. A revised method for detecting the lectin-like factor employs the serum IgM fraction, rather than whole serum, separated on an S-300 Sephacryl gel filtration column. Elevated lectin titers were found in 95 percent of 43 obligate heterozygotes, 89 percent of patients with cystic fibrosis, 64 percent of siblings of patients with cystic fibrosis, and 5 percent of 60 control subjects. Lectin activity was shown to be associated with the IgM fraction of serum and to result from the binding of a low-molecular-weight co-factor to IgM. Unbound cofactor was also detected in a serum fraction from 81 percent of the heterozygotes and 88 percent of the cystic fibrosis homozygotes, and in a urine fraction from nine patients with cystic fibrosis who had positive serum lectin activity, but not in any urine sample from heterozygous subjects. The presence of co-factor in the urine of cystic fibrosis homozygotes suggests higher serum levels of this agent that spill over into the urine. Intravenous antibiotic therapy removes the lectin and co-factor from blood and urine for variable periods of time, suggesting a beneficial effect of antibiotics in cystic fibrosis apart from their antibacterial action. Detection of the lectin and co-factor are useful in screening for cystic fibrosis carriers, although the somewhat subjective biologic assay requires much experience to achieve reliability.

Fractionation of rat liver glycogen particles on a sephacryl S-1000 gel column and their structural properties.

(1984). Fractionation of Rat Liver Glycogen Particles on a Sephacryl S-1000 Gel Column and Their Structural Properties. Agricultural and Biological Chemistry: Vol. 48, No. 1, pp. 215-217.

Purification of human alpha 1 antiprotease-pancreatic elastase complex. Interaction with homologous elastin.

A human alpha 1-antiprotease (alpha 1.AP)-human pancreatic elastase ( HPE11 ) complex was isolated from plasma, or prepared from commercial alpha 1.AP. The complex was identified and isolated by affinity chromatography, using Concanavalin A or IgG anti-elastase as ligands, in association with Sephacryl gel filtration. The alpha 1.AP- HPE11 complex was shown to bind with aorta and with purified aortic elastin. In both cases, the complex exhibited elastinolytic activity.

Clathrin-coated vesicles purified from a human B lymphocyte cell line contain newly synthesized glycoproteins.

Clathrin-coated vesicles were purified from the cultured human B lymphocyte cell line RPMI 1788. These preparations were shown to be substantially free of contaminating membranes by the following criteria: (a) polypeptide composition, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) electron microscopy, (c) lack of binding of intact vesicles to concanavalin A-Sepharose, (d) migration of coated vesicles as a single major peak when applied to a Sephacryl S-1000 gel filtration column. An association between HLA-A, -B, and -DR antigens and immunoglobulins with coated vesicle fractions was demonstrated by lentil lectin affinity chromatography and by immunoprecipitation using antisera directed against these glycoproteins. [35S]Methionine pulse-chase experiments revealed that there was a kinetic correlation between the expression of newly synthesized glycoproteins and their presence in coated vesicles. These results indicate that coated vesicles may be involved in the transport of cell surface and secreted eukaryotic glycoproteins through the cell to their final destinations at the plasma membrane or in the extra-cellular medium.

809 DETECTION AND CHARACTERIZATION OF ERYTHROPOIETIC INHIBITORS IN ANEMIA OF CHRONIC RENAL FAILURE

The mechanism of anemia in pts with end-stage renal disease was studied by assessing erythroid colony growth in methylcellulose cultures. Peripheral blood BFU-E from 10 anemic pts were normal when cultured in control serum (mean 22 BFU-E/105, range 14-41 vs control mean 18±10/105), but declined a mean of 67% when autologous uremic serum was substituted. Sera from 53 of 60 pts cultured with control marrow produced a mean decrease in BFU-E of 74% and in CFU-E of 79%. The serum inhibition was confirmed by reproducing the effect on control marrow colony growth with specific fractions of pts' serum separated by sephacryl gel chromatography. Neither peritoneal dialysis nor hemodialysis reduced the inhibitory activity, but it disappeared with successful renal transplantation. Analysis of uremic serum revealed a striking increase in a ribonuclease of M.W. 33,000 in all pts (9,500-40,000u/ml vs control mean 1,047±247u/ml) that was not eliminated by therapeutic dialysis. Purified ribonuclease produced dose-dependent inhibition of control marrow CFU-E but had unpredictable effects on BFU-E. We conclude that in anemic uremic pts: erythroid progenitors are adequate; the serum contains erythropoietic inhibitors; a ribonuclease is increased and appears to have a role in the erythropoietic suppression.

CHANGES IN GLOMERULAR CAPILLARY PERMEABILITY TO DEXTRANS (D) DURING DEVELOPMENT

An increase in the permeability of the glomerular capillaries has been suggested to contribute to the rise in GFR occurring during maturation. This is difficult to reconcile with the observation that the urine of the newborn contains more protein than that of the adult. It is this inconsistency that prompted us to measure the clearance of polydisperse tritiated D (Einstein-Stokes radius (r) 18-58A) in 6 newborn and 7 six-week old puppies. The separation of D according to molecular size was accomplished by fractionating samples of plasma and urine on a Sephacryl gel column. At both ages, the permeance of D molecules with radii less than 23A was similar to that of 125I iothalamate (r∼5A), while larger molecules were restricted to a progressively greater degree. The relative clearance of D ranging in size from 24-40A was 21 ± 1% (SE) greater in the newborn than in the more mature puppy. No significant differences between the two age groups were found in the relative clearances of D smaller than 23A or larger than 41A. Although these data are open to various interpretations, the simplest is that the number of pores per unit of glomerular capillary surface area is larger in the newborn than in the older pupppy. Electrical charge notwithstanding, the existence of a greater pore density would explain the presence of relatively large amounts of protein in the urine of the newborn, but it cannot be construed to account for the low GFR prevailing at early age.

Purification, properties, and kinetics of D-ribulokinase from Aerobacter aerogenes.

The enzyme D-ribulokinase from Aerobacter aerogenes was purified to near homogeneity. The molecular weight, as determined by Sephacryl gel chromatography, is 116,000. The subunit molecular weight, determined by sodium dodecyl sulfate-gel electrophoresis, is 59,000, suggesting that D-ribulokinase is a dimer of identical subunits. Initial rate kinetic studies, involving substrate analogs and products, were carried out. These investigations support a kinetic mechanism of the Random Bi Bi type. Isotope partitioning, utilizing D-[3H]ribulose, indicates that the mechanism is steady state Random Bi Bi.

Evidence for the presence of an antitumor factor in serum of normal animals

We have previously reported finding a factor with antitumor activity (TNF, tumor necrosis factor) in extracts of serum from normal mice. The possibility that TNF exists in the blood of normal animals of other species was explored. Horse, mouse, dog, human, sheep, calf, rat and shark serums were fractionated with (NH4)2SO4 and filtered through S-200 sephacryl gel. Proteins of molecular weight 90,000 to 180,000 were pooled, concentrated and dialyzed. TNF, determined by L-cell assay in vitro and Meth A assay in vivo found in fractions from mouse, dog and human serum. Agarose electrophoresis of the TNF from mouse and human serum indicated the principle components were alpha 1-alpha 2 globulins. Preparative PAGE indicated that mouse TNF migrated slowly and was made up of at least 4 components while human TNF was a faster moving, monomeric protein.