Ouchterlony Plates Research Articles

The road not taken or how I learned to love the liver: A personal perspective on hepatitis history

The road not taken or how I learned to love the liver: A personal perspective on hepatitis history

The unexpected outcomes of medical research: serendipity and the Australia antigenBlumberg BS, Alter HJ, Visnich S. A new antigen in leukemia sera [J Am Med Assoc 1965;191:541–546

The 'Australia Antigen' is found in the sera of some normal individuals from foreign populations. The total absence of the antigen from the sera of normal United States' subjects and its relatively high frequency in acute leukemia suggests that the presence of the antigen maybe of value in the diagnosis of early acute leukemia. Whether the antigen results from or precedes the leukemia process remains to be seen. (Abstract reproduced by permission of J Am Med Assoc 1965;191:541 - 546.) Many of the major discoveries in science have been the product of chance observations that were pursued by the curious mind to their plausible and provable explanation. Mendel's peas and, at least apocryphally, Newton's apple, come to mind as paradigm shifting observations that respectively opened the fields of genetics and established the laws of gravitation. Fleming's chance observation of the inhibition of bacterial growth in the presence of bread mold ultimately produced the first antibiotic. The unexpected link between the Australia antigen and the hepatitis B virus may be less encompassing than genetics or gravitation, but nonetheless is a classic example of a serendipitous observation pursued to major unexpected payoffs, including the prevention of post-transfusion hepatitis, the development of a vaccine, the prevention of hepatocellular carcinoma and laying the foundation for

Identification and Characterization of Aldose Reductase in Cultured Rat Mesangial Cells

Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.

The discovery of secretory IgA and the mucosal immune system

After completing my medical training, I began an affair with research that has not yet ended. Rockefeller University accepted me into their PhD program and, because of my interest in protein chemistry, I asked Dr Henry Kunkel to be my advisor. As was the tradition in the Kunkel lab, there were a large number of fellows and visiting scientists studying an array of medically related problems. One of the investigators, Halstead Holman, was interested in patients who had a gastrointestinal (GI) disease that was characterized by the transudation of large amounts of serum proteins into their GI tracts the so-called protein-losing enteropathies. During the course of these studies, Hal showed me an Ouchterlony plate from a normal control subject that contained GI fluid reacting with antiserum to several serum proteins. Macroscopically, by the intensity of the precipitation arc, the fluid seemed to contain much more gamma-globulin than albumin. This was surprising because by simple transudation, one would expect a larger proportion of the smaller albumin molecule. I suggested that, since the fluid was obtained via a nasogastric tube, these results might be due to swallowed saliva. None of us knew very much about the proteins present in saliva, and a few weeks later I simply expectorated into a beaker and, using gel-diffusion techniques with defined antisera, confirmed that human saliva gave a similar pattern to that of gastric fluid.

Detection of serum antibodies in human Hymenolepis infection by enzyme immunoassay

Although hymenolepiasis is the commonest cestode infection of man, there are no data available on the human immune response to this parasite. Thus, in order to determine if infection induces antibodies against Hymenolepis nana antigens, sera from 52 infected children were initially studied on Ouchterlony plates and then by enzyme-linked immunosorbent assay ( elisa), using a crude antigenic extract prepared from scolex and neck regions of adult worms. In addition, sera from persons with cysticercosis, taeniasis and other parasitoses, and normal human sera, were studied. Only one serum from the Hymenolepis group showed precipitin antibodies against H. nana antigen, while several were positive by elisa. The sensitivity of the elisa was 84·62% and its specificity was 100%. Very high cross-reactivity rates were obtained with taeniasis (70·6%) and cysticercosis (75%) sera. These results show that Hymenolepis infection in man induces a low but detectable humoral immune response. Although not useful for diagnostic purposes, this may be relevant to the serodiagnosis of other tissue cestode infections of man, since antibodies detected in serological tests used for the diagnosis of cysticercosis, and probably hydatidosis, could be induced by H. nana instead of Taenia solium or Echinococcus larvae.

Purification and characterization of cytochrome P450 isozymes from β-naphthoflavone-induced adult hen liver

Cytochromes P450 βNF-A, βNF-B, and βNF-C were purified from β-naphthoflavone-treated adult hens. Cytochrome P450 βNF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 βNF-A 1 and βNF-A 2 for property comparison. The cytochromes βNF-A 1 and βNF-A 2 were induced by both phenobarbital and β-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH 2-terminal amino acid sequence. However, P450 PB-A differed from βNF-A 1/βNF-A 2 in peptide pattern after partial proteolysis by α-chymotrypsin and Staphylococcus aureus V 8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from βNF-A 1/βNF-A 2, in that its antibodies cross-reacted with P-450 of normal, PB-, and β-NF-induced rabbit liver microsomes. The cytochromes βNF-B and βNF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH 2-terminal amino acid sequence. The most notable difference between βNF-B and βNF-C was that the anti-βNF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by β-NF-induced microsomes. These activities increased 40- to 50-fold in β-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of βNF-B and βNF-C were different from those of mammalian and other nonmammalian species.

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.

73 IS HPRT-LIKE PROTEIN PRESENT IN LESCH-NYHAN PATIENTS?

A complete deficiency of hypoxanthine-guanine phosphoribosyl-transferase (HPRT) activity results in the Lesch-Nyhan syndrome. Of the 15 Lesch-Nyhan patients studied by Wilson et al. (1), HPRT cross-reactinq material (CRM) was detected in lymphoblasts from only four (1.3%, 50%, 72% and 92% of control concentrations) whereas 12 had HPRT-specific mRNA. None of the three patients lacking mRNA had detectable CRM. We have studied CRM levels in haemolysates and lymphoblast lysates from two Lesch-Nyhan patients (R.W. and J.G). In the first series of experiments, normal CRM levels were measured and lysates from both patients gave strong precipitation lines in immunodiffusion analysis. In the second series, using a different HPRT preparation in the radioimmunoassay (RIA), very low CRM levels (0.5% and 0.1%) were obtained, although strong precipitation lines were still observed on Ouchterlony plates. A possible explanation for these discrepancies lies in a change in structure of normal HPRT which we have observed on storage, leading to a reduction in binding affinity for the antibody. The enzyme used in the first series of CRM determinations had been stored for an extended period, whereas enzyme used in the second series was freshly prepared. This explanation implies that the protein present in Lesch-Nyhan patients binds to the antibody with a lower affinity than does the normal enzyme (see also ref.2). These studies suggest that HPRT-like protein may be present in a higher percentage of Lesch-Nyhan patients than previously thought and may be present even when HPRT-specific mRNA cannot be detected, as in patient R.W. 1. Wilson, J.M., Stout, J.T., Palella, T.D., Davidson, B.L., Kelley, W.N. and Caskey, C.T. (1986) J.Clin.Invest. 77:188–195. 2. Wilson, J.M., Baugher, B.W., Landa. L. and Kelley, W.N. (1981) J.Biol.Chem. 256:10306-10312.

Immunoblot analysis of the circulating antigens occurring in serum of rats infected with Angiostrongylus cantonensis.

Following the infection of rats with Angiostrongylus cantonensis, the occurrence and molecular features of circulating antigens (CAs) were analyzed, with special reference to their origin in worms, by SDS-PAGE combined with an immunoblotting technique. Antisera against the CAs were obtained by immunizing rats with sera from rats 35, 91, and 150 days postinfection. The anti-sera, referred to as anti-CA(35), anti-CA(91), and anti-CA(150), respectively, formed one or two precipitin lines when tested in Ouchterlony plates against extracts of digestive organs (DE) and of reproductive organs (RE) from adult female worms. When the anti-CA(35) was used as a blotting antibody under nonreducing conditions, a set of clearly spaced narrow bands with molecular weights (mol. wt.) in the range of 90,000-180,000 daltons developed only in the case of the DE. Besides the antigen(s), additional bands with mol.wt. of 115,000 and 185,000 daltons were revealed in the case of the RE when two other antisera were used. Immunoblot analysis of the immunoprecipitates, derived from anti-CA(150) and sera of infected rats, revealed the occurrence of two types of protein as the major CAs: one had a mol.wt. in the range of 140,000-180,000 daltons and was found in the serum 14 days postinfection, and the other, with a mol.wt. of 185,000 daltons, was found in the serum 35-150 days postinfection. Immunohistochemical studies localized the CAs predominantly in the cytoplasm of both uterine eggs and maturing oocytes.

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake ( Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24 000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima position and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborted by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.

Purification of human leucocyte pyruvate kinase

The M 2 form of pyruvate kinase (M 2-PK) was purified from human leucocytes by a new method involving a succession of two different Dyematrex agarose chromatographies. The main step consisted of an orange dye affinity column with elution by fructose-1,6-diphosphate. This purification procedure allowed us to obtain M 2-PK with a specific activity of 433 I.U./mg of protein, i.e. a 188-fold purification with an overall yield of 33%. The homogeneity of this preparation was verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis and double immunodiffusion in Ouchterlony plates. Anti-M 2-PK antibodies obtained from rabbit neutralized the enzyme activity. Their specificity with regard to other types of PK showed that anti-M 2-PK also reacted with M 1-PK but not with R-PK.

Immunological studies on ?-amylase of Bacillus cereus

An antiserum against the β-amylase from Bacillus cereus BQ10-S1 Spo II was prepared using rabbits. The antiserum obtained was confirmed to form a specific immunoprecipitate with the purified β-amylase and showed a single band of protein with a molecular weight of 6.0x104 on the nitrocellulose sheet by the Western-Blotting method. The antiserum showed a precipitin line with the β-amylase from B. megaterium strain no. 32 by the Ouchterlony technique. However, the spur was formed on the Ouchterlony plate between the line of immunoprecipitin of the β-amylase from B. cereus BQ10-S1 Spo II and that from B. megaterium strain no. 32. On the other hand, no immune reaction occurred with the β-amylase from B. polymyxa no. 72 and those from higher plants such as soybean and barley.

Isolation and characterization of fibronectin from baboon plasma.

Fibronectin has been purified from baboon plasma by affinity chromatography on immobilized gelatin. Baboon fibronectin has subunit sizes, CNBr and thermolysin peptide patterns and an amino acid composition similar to human fibronectin. In Ouchterlony plates, baboon plasma fibronectin gives an immunological reaction of identity with human fibronectin, and a partial reaction with hamster fibronectin, using a goat antibody to human fibronectin. By radial immunodiffusion and rocket immunoelectrophoresis, using the same antibody, baboon fibronectin gives a dose response pattern similar to human fibronectin. Plasma concentrations of fibronectin in baboons are similar to those in humans. Fibronectin is thus another interesting protein that is closely related to its human counterpart and can be studied in subhuman primates using available antibodies to the human protein.

Benzo[ a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

We have purified five isozymes of liver microsomal (LM) P-450 from β-naphthoflavonefed rainbow trout. Four forms (LM 3, LM 1, LM 4a and LM x) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LM x into two components, LM 2 and LM 4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight ( M r ), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a λ max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[ a]pyrene in a reconstituted system. A second form, LM 4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm 4b by M r , λ max, or activity towards benzo[ a]pyrene. Form LM 2 (2% yield) had a specific content of 10.8, a M r of 54,000, a λ max of 449.5 nm, and was not effective in reconstitution of benzo[ a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, lm 1 and LM 3. Neither lm 1 nor LM 3 was active towards benzo[ a]pyrene. The properties of LM 2, LM 4a and LM 4b, were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM 4a and LM 4b, each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM 2, and LM 2-IgG did not cross-react with any other purified P-450. Benzo[ a]pyrene-hydroxylase, catalyzed by either LM 4a or LM 4b, was inhibited by LM 4b-IgG but not by LM 4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM 4a and LM 4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to α-naphthoflavone, and regioselectivity towards benzo[ a]-pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.

Cytochrome P-450 isozymes in salmonids determined with antibodies to purified forms of P-450 from rainbow trout

Abstract Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM4b)∗ appears to be a P-448 type of cytochrome. A minor form (LM4a), having properties very similar to LM4b, was also obtained. In addition, a P-450 form (LM2) was isolated, with properties quite different from LM4a or LM4b, including a high rate of aflatoxin B1 (AFB1) metabolism (Williams & Buhler, 1983c). Antibodies to all three forms were obtained from rabbits. The IgGs prepared against LM4a and LM4b both cross-reacted (forming lines of identity) equally well with both antigens on Ouchterlony plates. Rat P-448 cross-reacted (without lines of identity) with both LM4a- and LM4b-IgG. LM4b-IgG was much more effective than LM4a-IgG for inhibition of LM4a or LM4b reconstituted benzo[a]pyrene (BP) hydroxylase, suggesting that these two antibodies recognize different antigenic sites. The LM2-IgG did not cross-react with any of the other rat or trout cytochromes P-450 examined. Levels of LM2 and LM4b in microsomes from untreated, polychlorinated biphenyl (PCB), phenobarbital (PB) or BNF-treated trout were estimated with an immunological technique involving electrophoresis on SDS-PAGE, followed by transfer to nitrocellulose and staining with either LM 2 - or LM4b -IgG. The ratio of LM 4b LM 2 in microsomes from PCB- or BNF-treated rainbow trout was much higher than 1, whereas the reverse was true with microsomes from untreated rainbow trout. These results are consistent with previous observations (Vodicnik et al., 1982) that pretreatment with BNF induced the synthesis of a P-448 type cyytochrome, presumably responsible for the great increase in the metabolism and activation of BP seen in these fish. Conversely, pretreatment with PB did not affect the levels of either LM2 or LM4b. This specific immunological technique should make it possible to assay the levels of these P-450 and P-448 isozymes in various strains of rainbow trout and other species of fish. In addition, the effect of age, sex, diet and exposure to P-450 and P-448 inducers could be examined and, perhaps, utilized to predict the relative risk of certain populations to pollutants activated by these different isozymes.

Purification and comparative properties of NADPH-cytochrome P-450 reductase from rat and rainbow trout: Differences in temperature optima between reconstituted and microsomal trout enzymes

NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of β-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin- O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat.

Altered cytochrome P-450 in a yeast mutant blocked in demethylating C-32 of lanosterol.

Spectroscopic and enzymatic analysis of a Saccharomyces cerevisiae mutant in sterol biosynthesis (SG1 (erg 11); Trocha, P. J., Jasne, S. J., and Sprinson, D. B. (1977) Biochemistry 16, 4721-4726) that was blocked in demethylating C-32 of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) showed that it contained low levels of cytochromes P-450 and b5 compared to those present in the parent strain D-587, while NADPH-cytochrome c reductase activity was elevated. The fungicide buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), which bound specifically to yeast cytochrome P450 responsible for demethylating C-32 of lanosterol and effected a Type II spectral change, did not react with SG1 cytochrome P-450. On the other hand, cholate-solubilized microsomes from SG1 formed a single precipitin line on Ouchterlony plates with antibodies raised against purified (Yoshida, Y., Aoyama, Y., Kumaoka, H., and Kubota, S. (1977) Biochem. Biophys. Res. Commun. 78, 1005-1010) yeast cytochrome P-450 specific for demethylating C-32 of lanosterol. Hence, mutant SG1 contained an altered protein which retained the antigenicity of cytochrome P-450 responsible for demethylating C-32 of lanosterol but lost its catalytic activity.

Purification of human erythrocyte phosphoglycerate kinase by dye ligand affinity chromatography

A new method for the purification of human erythrocyte phosphoglycerate-kinase involving affinity chromatography on dye-ligand media (Red A), in the presence of 3-phosphoglycerate and ATP, is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate, by amino acid analysis and by immunoprecipitation in Ouchterlony plates. Peptide mapping of tryptic digests of the purified enzyme was performed and the immunoneutralization of the enzyme activity evaluated with rabbit antibodies.

Partial characterization of the inactive mutant form of human red cell bisphosphoglyceromutase and comparison with an alkylated form

The trifunctional enzyme bisphosphoglyceromutase (or diphosphoglycerate mutase) (EC 2.7.5.4) was purified from human red cells and injected into two chickens. Specific anti-bisphosphoglyceromutase antibodies were produced that displayed a single precipitation line on Ouchterlony plates and on immunoelectrophoresis. No cross-reaction of these antibodies was detected with phosphoglyceromutase, the common glycolytic enzyme. Immunoneutralization of bisphosphoglyceromutase and of its two other activities, i.e., bisphosphoglycerate phosphatase and phosphoglyceromutase, was observed for a purified preparation. The anti-bisphosphoglyceromutase antibody reacts with the inactive enzyme present in the hemolysate of a mutant human subject. It also binds bisphosphoglyceromutase inactivated by N-ethylmaleimide, a strong alkylating agent of SH groups. Active bisphosphoglyceromutase is stable at 55°C, whereas the inactive forms of the mutant and of the alkylated hemolysates are thermolabile. These forms can be protected against thermal precipitation by 4 mM 2,3-diphosphoglycerate and 4 mM 3-phosphoglycerate. These findings afford evidence that the binding of the substrates on the bisphosphoglyceromutase molecule is not prevented by alkylation nor by the mutation of the hereditary inactive enzyme.

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.