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Browning reactions of amino sugars were observed in a variety of sterile pH buffers at 25-37 degrees C. These reactions were signaled by an increase in absorbance at 273 nm, followed by an increase in absorbance at 320-360 nm. The reactions were maximal at pH 7.0 in phosphate buffer. Acidic solutions (pH less than 2.2) of 50 mM D-glucosamine hydrochloride gave only a negligible reaction and 2-acetamido-2-deoxy-D-glucose was unreactive. Half of the D-glucosamine in a 100 mM solution in sterile 0.2 M sodium phosphate buffer, pH 7.4, at 37 degrees C decomposed or was transformed in 27 h. A comparison of reactivity in generating A273 and A340 chromophores showed D-mannosamine greater than D-galactosamine greater than D-glucosamine. Permanganate oxidation of incubated glucosamine solutions afforded a compound which chromatographed like 2,5-pyrazinedicarboxylic acid and gave the same ultraviolet absorption spectrum. This, together with fractionating and thin-layer chromatography of the products of glucosamine incubation, suggests that 2,5-bis(tetrahydroxybutyl)pyrazine is formed as one of the products of autocondensation of D-glucosamine in accord with the report of Candiano et al. (1988, Carbohydr. Res. 184, 67-75) on products formed in glucosamine-lysine incubation mixtures. Formation of products absorbing at 325-360 nm was inhibited by the chelator diethylene-triaminepentaacetic acid. This suggests that the later reactions may be mediated by a metal-stimulated free radical mechanism. After 4 days incubation high molecular weight products with absorbance maxima at 273 nm and 325-360 nm were detected. Some of these were retained by dialysis membranes of molecular weight cut-off greater than 3500 and greater than 12,000.

We have developed a new method of preparing tissues for analysis of thyroid hormones and metabolites which eliminates troublesome lipids and proteins. Frozen tissue is homogenized by grinding with dry ice in a Waring blender, and the moist powder obtained is extracted with chloroform:methanol (2:1). In a modification of the Folch procedure for the total separation of lipids, the filtered extracts then are partitioned into polar and nonpolar layers by the addition of 0.05% aqueous calcium chloride. The upper phase contains the iodocompounds of interest as well as all salts and small polar molecules. The lipids remain dissolved in the lower phase after it is back-extracted with pure upper phase. The combined upper or aqueous methanol layers are lyophilized and the residue is taken up in methanol to yield a concentrated solution ready for analysis of iodocompounds. The greater clarity of the extract permits application of larger samples for two-dimensional paper chromatography than has been customary. For gradient analysis by reverse-phase ion-pair high-performance liquid chromatography (HPLC), the nitrogen-evaporated methanol extract is dissolved in the initial mobile phase, 0.1% H 3 PO 4 , for injection onto the column. Using these methods we have achieved the first reported separation of 125 I-labeled tissue iodothyronine metabolites by HPLC. The new method of extraction is of general applicability to any biological sample which might be analyzed in thyroid hormone metabolism research.

Rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and the amplitude of the diurnal variation of this enzyme are progressively reduced to very low levels within 1 week after the onset of diabetes induced by streptozotocin. Daily insulin therapy to 7-day diabetic rats restores the activity and the amplitude of this diurnal variation in enzyme activity to near-normal levels within 4 days. Insulin also produces a rapid 2-hr stimulation of the reductase activity in diabetic rats to the level found in normal animals at that time of day regardless of the duration of diabetes. Hence, insulin is required for the diurnal rise of reductase activity in rat liver. Glucagon, dibutyryl cyclic AMP, and hydrocortisone, in contrast, markedly inhibit the diurnal rise of reductase activity in normal rats. Therefore, the relative concentrations of insulin, glucagon, and glucocorticoids are important in the regulation of the diurnal variation of hepatic reductase activity.

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of 14C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-3H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway. Fractionation data of cells grown on [3H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the 3H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.

A sensitive assay has been devised for glutathione- S -arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [ 35 S]glutathione. Activity of the order of 2–3 nmoles of conjugate formed during a 5-min incubation can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione- S -arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and two or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. Activities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks. The sheep liver transferase required neither metal ions nor cofactors such as FAD, pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of the transferase has been estimated to be about 40,000. K m values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 m m , respectively. K m values for glutathione decreased with increasing pH, whereas the K m values for naphthalene-1,2-oxide were independent of pH in the range of 6.5–8.

Uric acid has been proposed to be an important antioxidant and free radical scavenger in humans. Of the purine and pyrimidine compounds examined in this study, uric acid showed the greatest susceptibility to ozone-induced degradation. The parent compounds, purine and pyrimidine, were more resistant to ozonation than were the nucleobases. When the degradation of OH-substituted purines was examined, it was found that the more OH groups on the purine ring, the more readily the purine was degraded. Urea and allantoin were identified as degradation products of uric acid. The relative rates of nucleobase degradation in the presence and absence of uric acid were compared. Uric acid protected thymine, guanine, and uracil from degradation by ozone. In this system uric acid was found to protect the nucleobases as effectively as reduced glutathione.

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [14C]cytidine nucleotides to [14C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [3H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [3H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.

The biosynthesis of lipid-linked oligosaccharides has been studied in first trimester human placentas. Tissue was pulsed with [2-3H]mannose, [1-3H]glucosamine, and [1-3H]galactose (for [3H]glucose incorporation). The lipid-linked oligosaccharides (OSL) were purified on DEAE-cellulose. After cleaving the lipid from OSL the oligosaccharides were purified by paper chromatography and borate high-voltage electrophoresis. Four major oligosaccharides with the composition Glc(0-3)Man9GlcNAc2 thus obtained were characterized enzymatically by digestion with endo-beta-N-acetylglucosaminidase H and alpha-mannosidase, and chemically by methylation, acetolysis, and Smith degradation. While identical Glc(1-3)Man9GlcNAc2 oligosaccharides have been isolated from other in vivo systems, the presence of Man9Glc2 is novel for human placenta. Furthermore, Man9GlcNAc2 is present in appreciable amounts in placenta. Second, one mannose in Man9GlcNAc2 had a significantly higher specific radioactivity than the other mannosyl residues of the Glc(0-3)Man9GlcNAc2 oligosaccharides. Third, the differential labeling in Man9GlcNAc2 and our pulse-chase studies indicate that Man9GlcNAc2 is not a major precursor of Glc3Man9GlcNAc2. The data also suggest that the ninth mannose, which has the highest radioactivity, may be incorporated in a different subcellular compartment and may serve as a regulatory step in the biosynthesis of lipid-linked oligosaccharides. A model is proposed which outlines possible multiple pathways of lipid-linked oligosaccharide biosynthesis in human placenta.

The data presented here describe new findings related to the bioconversion of adenosine to 9-β- d -arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2′- 18 O]- and [U- 14 C]adenosine, the 18 O: 14 C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2′ hydroxyl of adenosine to ara-A with retention of the 18 O at C-2′. In experiments with [3′- 18 O]- and [U- 14 C]-adenosine, [U- 14 C]ara-A was isolated; however, the 18 O at C-3′ is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18 O: 14 C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or d -ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U- 14 C]adenosine, there was a 9.9% conversion to [U- 14 C]ara-A; with [2′- 3 H]-adenosine, there was a 8.9% release of the C-2′ tritium from [2′- 3 H]adenosine which was recovered as 3 H 2 O. Third, the release of 3 H as 3 H 2 O from [2′- 3 H]adenosine was confirmed by incubations of the enzyme with 3 H 2 O and adenosine. Ninety percent of the tritium incorporated into the d -arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a K m of 8 × 10 −4 M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2′ hydroxyl of the nucleoside antibiotic, tubercidin to form 7-β- d -arabinofuranosyl-4-aminopyrrolo[2,3- d ]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3′-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.

Teichuronic acid, the Micrococcus luteus cell wall polysaccharide which consists of D-glucose and N-acetyl-D-mannosaminuronic acid, is synthesized in vitro from uridine diphosphate N-acetyl-D-glucosamine, uridine diphosphate N-acetyl-D-mannosaminuronic acid, and uridine diphosphate D-glucose in a series of reactions catalyzed by a particulate enzyme preparation. Several lipid-linked intermediates are formed, of which the first three are called components A, B, and C. The formation of these intermediates is inhibited by tunicamycin. The lipid moiety of the intermediates is approximately 95% undecaprenol and 5% dodecaprenol as determined by mass spectrometry. The oligosaccharide moieties of components B and C are the disaccharide, N-acetyl-D-mannosaminuronyl-(1,3)-N-acetyl-D-glucosamine, and the trisaccharide, N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminuronyl++ +-(1, 3)-N-acetyl-D-glucosamine, respectively, as determined by the complete degradation of the former and partial degradation of the latter by the alkaline beta-elimination reaction. The saccharide and lipid moieties of the intermediates are linked through pyrophosphate. Thus, component A is P1-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, component B is P1-N-acetyl-D-mannosaminuronyl-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, and component C is P1-N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminurony l-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate.

A sugar, previously unobserved, was formed from cellobiose by enzymes in the supernatant fraction of Ruminococcus albus cultures. The sugar was isolated by preparative paper chromatography and was subsequently identified by means of its melting point, optical rotation, infrared spectrum, and chromatographie behavior as 4-O-β- d -glucopyranosyl- d -mannose. The epimerase was assayed by determining the conversion of cellobiose into glucosylmannose by means of quantitative paper chromatography. Maximum activity occurs near pH 7.0. The enzyme is unstable at room temperature and loses activity rapidly above 38 °. At equilibrium, the enzymic interconversion favors the cellobiose configuration. The reversible epimerization of cellobiose and glucosylmannose apparently occurs through a direct reaction at carbon-2 of the reducing moiety of the disaccharide.

Summary o 1. Methods for the detection of steroids in paper chromatography have been described and evaluated for 31 compounds. 2. A new method of detection of steroids involving phosphomolybdic acid has been reported. 3. A method using silicotungstic acid has been extended to cover many new compounds.

Radioactive glucosamine is incorporated into the outer segments of the rod cells of bovine retinas incubated in vitro . One component of the outer segment labeled in this process is rhodopsin which can be extracted with detergent, purified by sequential chromatography on calcium phosphate-Celite and agarose, and shown to be light sensitive by its altered chromatographic mobility. The radioactive component can be released from rhodopsin by acid hydrolysis and shown to migrate with glucosamine on paper chromatography. In double label experiments both glucosamine and leucine are incorporated into rhodopsin. The time course of glucosamine incorporation is similar to that of leucine. The system supports prolonged synthesis of both the polypeptide and oligosaccharide portions of the rhodopsin molecule in vitro .

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA2, globoside, and GM2. Hexosaminidase A and B had similar ability to catalyze the hydrolysis of synthetic N-acetylglycosaminides. The hexosaminidase B from the liver of a Tay-Sachs disease patient had a slightly higher Michaelis constant toward the same substrates as the control hexosaminidase B. Radioactively labeled GA2, N-acetyl-[1-14C] galactosaminyl (β 1 → 4)-galactosyl (β 1 → 4)-glucosyl (β 1 → 1)-ceramide was prepared biosynthetically. Hexosaminidase A and B from normal liver had identical Michaelis constants for the hydrolysis of GA2; whereas, the hexosaminidase B from a Tay-Sachs disease patient possessed a higher Michaelis constant. Globoside was hydrolyzed by both hexosaminidase A and B, although hexosaminidase B has a significantly lower Michaelis constant than the hexosaminidase A. Under the conditions of incubation neither hexosaminidase A nor B catalyzed the hydrolysis of the N-acetylgalactosaminyl residue from ganglioside GM2. However, in the presence of a neuraminidase preparation from Clostridium perfringens plus hexosaminidase A or B at pH 3.8 there was significant enzymatic hydrolysis of GM2 to ceramide lactose. Both GM2 and globoside were found to be excellent competitive inhibitors of GA2 hydrolysis by hexosaminidase B. These results are discussed with respect to the accumulation of these glycolipids in two GM2 ganglioside-storage diseases.

N-Terminal analysis of chicken heart H 4 lactate dehydrogenase demonstrated the absence of a free amino acid or pyrrolidone carboxylic acid residue at the amino terminus of the protein. Quantitative determination of bound acetyl groups gave a value of 4.9 mole/mole of protein (mol wt of 135,000). The N-terminal peptide, characterized as N -acetyl-alanylthreonine, was isolated from pronase digests of the protein, and quantitative recovery experiments indicate that nearly all acetyl residues are found at the amino termini of the protein. The presence of leucinc as the C-terminal amino acid was confirmed using a tritium exchange method.

A spectrophotofluorometric assay was used to measure tryptophan oxygenase activity in several species. The fluorescent assay depends on the conversion of the product of the reaction, N-formyl- l -kynurenine, to anthranilate by means of the coupling enzymes kynurenine formamidase and kynureninase. These enzymes are easily obtained from l -tryptophan-induced N. crassa; and the product, anthranilate, is readily separated by organic extraction from other tryptophan catabolites and easily identified fluorometrically. With this assay, tryptophan oxygenase has been demonstrated in vitro for the first time in N. crassa.

The hydroxylation of a variety of specifically deuterated aromatic substrates by liver microsomes has been investigated. Labeled substrates which cannot readily ionize by loss of a proton from the ring substituent display deuterium retentions varying from 40 to 64% upon hydroxylation at the ring position bearing the isotopic label. These substrates include compounds with either electron-donating or electron attracting substituents (anisole-4- 2 H, diphenyl ether-4- 2 H, biphenyl-4- 2 H, N -methyl- N -(phenyl-4- 2 H)-benzenesulfonamide, toluene-4- 2 H, fluorobenzene-4- 2 H, chlorobromobenzene-4- 2 H, benzonitrile-4- 2 H, benzamide-4- 2 H, and nitrobenzene-4- 2 H). Substrates that can, however, ionize by loss of a proton from the ring substituent exhibit lower retentions ranging from 0 to 30% at pH 8. This class of compounds includes phenols such as salicylic-5- 2 H acid, aniline-4- 2 H, N -(phenyl-4- 2 H)-benzenesulfonamide, and various N -acylanilines-4- 2 H. As the acidity of the amide hydrogen increases in the acylanilines, migration and retention of deuterium decreases. A mechanism is presented which accommodates the retention values observed for both classes of substrates. The degree of retention for acetanilide-4- 2 H is strongly dependent upon the pH of the incubating medium ranging from 21 (pH 10) to 44% (pH 6). N -(Phenyl-4- 2 H)-benzamide shows a similar pH dependence whereas nonionizable substrates (biphenyl-4- 2 H or anisole-4- 2 H) do not display this dependence. The relative reactivity of the substrates studied toward the microsomal hydroxylating system correlates well with their reactivity toward chemical reagents which cause electrophilic substitution: i.e., reactive or electron-rich rings act as the best substrates. In chemical terms, the microsomal hydroxylating system acts as though it is a weak, selective electrophile.

An extracellular α-glucan, pullulan, elaborated by a strain of Pullularia pullulans , contains 0.6% of a maltotetraose subunit, as well as the already known major component, maltotriose. The majority, at least, of the maltotetraose is contained within the polymeric chains and is linked, through its terminal glucose units, by α-1→6— bonds.

Subcellular particles were isolated from the phosphorus-rich portion of rice bran using a differential centrifugation in nonaqueous media. Isolated particles were characterized by high contents of phosphorus and metals and low contents of proteins. Most of the phosphorus was found as phytic acid. Chemical composition and electron microscopic observation confirmed that isolated particles were derived from the aleurone layer and were distinguished from protein bodies.

The kinetic properties of gluconokinase from a pseudomonad have been examined. The catalytic reaction appears to follow a sequential (probably random) order of addition of substrates to the enzyme. The enzyme is highly specific for both ATP and gluconate. The Michaelis constants for ATP and gluconate are 1.04 × 10−3 m and 1.16 × 10−4 m , respectively. There appears to be a secondary site, distinct from the active site, at which ADP interacts with the enzyme. Although the overall reaction rate is lowered by the presence of ADP, this inhibition is compensated by a decrease in Km for both substrates in the presence of ADP. The nature of the ADP inhibition is pH dependent. At pH 6.5, the inhibition is mixed with respect to ATP and at pH 8.5, ADP behaves as a competitive inhibitor. At pH 7.5, the type of inhibition appears to depend on the concentration of the inhibitor. At low ADP concentrations, the inhibition appears to be competitive and at higher ADP concentrations, it is mixed. The ADP inhibition is relatively specific. Both dADP and IDP offer some inhibition, but both are weaker inhibitors than ADP. The properties of the enzyme are discussed in terms of the regulation of gluconate metabolism in the pseudomonad.

Three fractions, F-IA, F-IB, and F-II, have been obtained from extracts of Es-cherichia coli W by chromatography on DEAE-Sephadex. The guanosine 5′-triphosphate (GTP)-dependent transfer of aminoacyl-RNA to ribosomes is catalyzed by either F-IA or F-IB, and polypeptide synthesis is catalyzed by either fraction in the presence of F-II. Fraction F-IB is more labile than F-IA. Both F-IA and F-IB interact with GTP to form guanine nucleotide-enzyme complexes that are retained by Millipore filters. In the presence of aminoacyl-RNA, F-IA and F-IB interact with GTP to form a complex that is not retained by a Millipore filter. Guanosine 5′-diphosphate (GDP) also interacts with F-IA or F-IB to form a complex; however, no subsequent interaction with aminoacyl-RNA is observed. Both the GTP-dependent enzymic transfer of aminoacyl-RNA to ribosomes and the interaction of enzyme with GTP are inhibited by GDP. The enzymic transfer of aminoacyl-RNA to ribosomes is stimulated by NH4+ and a sulfhydryl compound, and the interaction of the guanine nucleotide-enzyme complex with aminoacyl-RNA is also stimulated by NH4+. Deacylated sRNA, which inhibits nonenzymic binding, has no significant effect on the enzymic binding of aminoacyl-RNA to ribosomes. The guanine nucleotide-aminoacyl-RNA complex formed by the interaction of GTP, phenylalanyl-RNA, and either F-IA, F-IB, or a mixture of the two can be recovered by filtration through a Millipore filter and chromatography on Sephadex G-25. The active fractions from the Sephadex G-25 column contain close to stoichiometric amounts of phenylalanine and the guanine moiety and the -γ-phosphate moiety of the GTP. The amount of phenylalanine transferred to the ribosomes from the complex is 2-fold greater than the amount transferred from phenylalanyl-RNA alone and is equivalent to the amount transferred from phenylalanyl-RNA in the presence of enzyme and GTP. The guanine moiety of the complex is also transferred to the ribosomes, but no significant transfer of the γ-phosphate of the GTP present in the active fractions is observed.

The presence of xylose-containing polysaccharide proteins in human brain (reported in 1962 by this laboratory) was confirmed by the isolation of xylose by partition chromatography and the preparation of xylose tetraacetate. Brain xylose is the d -isomer as indicated by its oxidation to xylonic acid with d -glucose oxidase. In protease-digested brain extracts, xylose and arabinose are present as constituents of acidic mucopolysaccharide peptides which can be precipitated by cetylpyridinium bromide (CPBr). An electrophoretically homogeneous mucopolysaccharide peptide was extracted from the CPBr precipitate with dilute acetic acid; it was strongly levorotatory, had the anodic mobility and the infrared spectrum of chondroitin 4-sulfate, and contained xylose, galactose, galactosamine, hexuronic acid, sulfate, and nonhexosamine N in the molar ratio, 1:2:7:9:8:2. Fractionation of the less acidic mucopolysaccharide peptides was achieved only after careful removal of basic proteins (by IR-120, H+) and glycoproteins (by reprecipitation with CPBr). The CPBr precipitate was dissolved in NaCl solution and reprecipitated with ethanol. Traces of residual glycoproteins were removed by gel filtration and the mucopolysaccharide-peptides were fractionated on AG1-X2 with NaCl. The fraction which was eluted with 0.8 n NaCl was homogeneous electrophoretically, had the electrophoretic mobility and the infrared spectrum of hyaluronate, and was digestible by hyaluronidase. It contained 43.7% hexuronic acid and 42.6% glucosamine; the hydrolyzate gave on paper chromatography arabinose and a single ninhydrin spot with a mobility slightly lower than that of glycine. Fractions eluted with 1 and 1.25 n NaCl contained mucopolysaccharide peptides with an electrophoretic mobility slightly higher than that of hyaluronic acid. Elution with 1.5, 1.6, and 2 n NaCl gave fast moving components, one of which coincided with chondroitin sulfate. Hydrolyzates of these fractions contained several hexuronic acids, glucosamine, and galactosamine; xylose and galactose were present. Serine and dicarboxylic amino acids were the dominant amino acids.

Studies on the isolation and characterization of DNA from yeast mitochondria (Mt-DNA)4 have been extended to mitochondria from respiratory deficient cells (ϱ− petites). Under appropriate conditions assuring the absence of gluocse repression such cells have been found to contain mitochondrial DNA in amounts comparable to those of the wild type. Mt-DNA from a neutral petitite exhibits a buoyant density in CsCl and a thermal transition midpoint (Tm) very close to that of its isogenic wild type. Mt-DNA's from two other isogenic ϱ− strains, known to be 10 and 50% suppressive respectively, show a very sharp thermal transition with a Tm of 66.5 ° in 0.15 m NaCl—0.015 m sodium citrate. Upon quick cooling almost complete renaturation is observed, as evidenced by second-cycle heating, again with a sharp transition and a Tm of 66.5 °. The average value of the buoyant density of this DNA in CsCl (at 20 °) is 1.6685 which is 0.003 g/ml lower than that reported for synthetic or crab d(A — T). There is virtually no change in the density of this Mt-DNA upon heating and quenching, with or without prior exposure to proteases, again confirming the great ease of its renaturation. In alkaline CsCl its density increases by 0.076 g/ml, indicating strand separation, but upon reneutralization the density returns to that of native Mt-DNA. Ready renaturation, as measured by thermal profiles, is found even for partially degraded Mt-DNA molecules. All these observations suggest that ease of renaturation is due to compositional homogeneity and therefore does not require these DNA's to exist as covalently continuous circles. The composition and homogeneity of the Mt-DNA from one of these suppressive strains (D310-2A-184) was confirmed by direct analysis of base composition: It was found to contain an equimolar amount of G and C as well as of A and T with the former two bases accounting for no more than four mole percent. The implications of these findings with regard to the mutagenic events reponsible for the formation of these aberrant DNA's and their possible genetic capabilities are discussed.

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.

The reaction on paper between reducing sugars and ammonia has been studied. The reaction products were separated by developing the paper chromatogram with an ammonia-containing solvent. In all cases there were obtained three ninhydrin-, as well as p-anisidine-, reactive spots, the relative intensities of which depended on the reaction time, on the concentration of ammonia, and on the presence or absence of catalysts. These spots are provisionally identified as hexose-ammonia, hexose-1-amine, and dihexosamine. The value of paper chromatography in study reactions involving labile intermediates is discussed.

An apparatus was developed for the external generation of singlet ( 1 Δg) molecular oxygen (singlet oxygen) for introduction into aqueous solutions of ribonucleosides and sodium ribonucleate (yeast). In contrast to previous conclusions on the specificity of singlet oxygen toward guanine residues, all ribonucleosides were reactive. The relative reactivities for guanine, uridine, cytidine, and adenosine were 26:13:8:1. Singlet oxygen was also found to react with sodium ribonucleate (yeast).

The metabolism of d - and l -lysine has been studied in the chicken using both 14C and 15N labels. In contrast to the rat, the chicken actively metabolized d -lysine and l -pipecolate. The data indicate that d -lysine is degraded via conversion to pipecolate, α-aminoadipate, α-ketoadipate, and eventually to CO2. The α-nitrogen atom of d -lysine is the first one removed from the carbon skeleton by this route. l -Lysine is metabolized by two alternate pathways which converge at α-aminoadipate. The major pathway, which results in the retention of the α-nitrogen atom of l -lysine in α-aminoadipate, includes saccharopine as a precursor of α-aminoadipate and is thus identical to l -lysine catabolism in rats. The pathway which is indicated to be the minor one includes l -pipecolate as a precursor of α-aminoadipate and results in the retention of the ϵ-nitrogen atom of l -lysine in α-aminoadipate. Even though saccharopine is indicated to be an intermediate in l -lysine breakdown, a large percentage of an intramuscularly injected dose is excreted unchanged in the urine.

Two soluble fucosyltransferases have been demonstrated in human serum. One enzyme transfers l -fucose from GDP- l -fucose to the terminal galactose residues of lactose, N-acetyllactosamine, and sialidase-treated α1-acid glycoprotein, to form the blood group H determinant, α- l -fucosyl-(1 → 2)-β- d -galactosyl-R. The second enzyme transfers fucose to the terminal N-acetylglucosamine residue of sialidase-, β-galactosidase-treated α1-acid glycoprotein. Serum from a donor with the rare “Bombay” Oh blood group (genotype hh) cannot transfer fucose to terminal galactose residues but has normal levels of the enzyme acting on sialidase-, β-galactosidase-treated α1-acid glycoprotein. This observation, as well as mixed substrate experiments, demonstrate that the two fucosyltransferase activities are due to two separate enzymes. The GDP- l -fucose:galactoside fucosyltransferase has a pH optimum of 5.5 and the following Km values: lactose, 31 m m ; N-acetyllactosamine, 7.5 m m ; sialidase-treated α1-acid glycoprotein, 6.4 m m . The GDP- l -fucose: N-acetylglucosaminide fucosyltransferase has a pH optimum of 5.0 and a Km for sialidase-, β-galactosidase-treated α1-acid glycoprotein of 1.2 m m . The serum GDP- l -fucose: N-acetylglucosaminide fucosyltransferase is distinct from the blood group Lewis-dependent enzyme in milk since the serum enzyme is present in serum from Le (a-b-)donors and since the Le-dependent fucosyltransferase could not be demonstrated in serum from donors carrying the Le gene.

The extracellular slime polysaccharide of Alcaligenes faecalis var. myxogenes strain 10C3 was isolated, purified, and shown to contain succinic acid (ca. 10%), glucose (ca. 70–80%), and small amounts of galactose and mannose. It seems to have β-glycosidic linkages. The polysaccharides obtained from cultures grown on glucose and on ethyleneglycol were studied. The two polymers had similar chemical and physical properties except that the polymer from glucose contained rather less mannose and had a higher specific viscosity than that from ethyleneglycol.

A particulate enzyme preparation obtained from rat liver catalyzes the transfer of glucosamine from UDP- N -acetyl- 14 C-glucosamine to endogenous protein acceptor(s). This transfer activity is found to be much higher in smooth microsomes than in rough microsomes. Unlabeled GDP-mannose stimulates incorporation, while other sugar nucleotides tested do not. The endogenous acceptor appears to be compartmentalized within the microsomes, but can be released by treatment with detergents or ultrasonic vibrations. Pronase digestion converts the products of this reaction from trichloroacetic acid-insoluble proteins to trichloroacetic acid-soluble glycopeptides. The radioactive label is incorporated as glucosamine, as identified by cochromatography on paper with authentic glucosamine, following acid hydrolysis of pronase glycopeptides.

For the E. coli maltodextrin phosphorylase system, the minimum normal primer, which permits indefinite elongation of the maltodextrin chain, is G 4 . However, B 4 acts as a pseudo-primer. B 4 plus G * -1-P produced slow B 5 * , but only traces of higher homologs. The newly synthesized slow B 5 * was 6 3 -α-maltosyl * maltotriose. Unlabeled slow B 5 was phosphorolyzed to radioactive G-1-P * in presence of 32 P i . The fact that G 4 is the minimum linear primer indicates that E. coli maltodextrin phosphorylase has four primer binding positions, I–IV (starting the numbering from the reducing end). Since B 4 can act as a pseudo-primer, position III can accommodate either a 4- or a 6-linked glucose unit. Models of B 4 and G 4 can be superimposed except at the third glucose unit from the reducing end. Hence, B 4 can bind at the phosphorylase primer site placing glucose units 1,2, and 4 at positions normally occupied by glucose units 1, 2, and 4 of G 4 .

An enzyme system obtained from light-grown cultures of the green alga, Chlorella pyrenoidosa, is shown to catalyze the reduction and epimerization of uridine diphosphate d -glucose to a compound characterized as uridiue diphosphate l -rhamnose; TPNH is required in the reaction. Uridine diphosphate d -galactose and thymidine diphosphate d -glucose participate to 75 and 25% the extent of uridine diphosphate d -glucose. Evidence is presented that the expected intermediate, uridine diphosphate 4-keto-6-deoxy- d -glucose, is produced in the interconversion of the two sugar nucleotides.

The oxidation of both N1-methylnicotinamide and pyridoxal appears to be catalyzed by a single liver enzyme, aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC 1.2.3.1). This notion is based on the following evidence: (1) Inbred strains of mice having characteristic and different levels of N1-methylnicotinamide oxidase activity also differ in their abilities to oxidize pyridoxal; the levels of the two activities have been found to vary among the strains in a parallel fashion. (2) The Km values of the N1-methylnicotinamide oxidizing activity for N1-methylnicotinamide and pyridoxal oxidizing activity for pyridoxal also vary among the inbred strains of mice in a parallel fashion. (3) The genetic determinants of pyridoxal and N1-methylnicotinamide oxidizing activity in the mouse do not segregate in the F2 generation or in backcrosses to the parental strains. (4) The levels of N1-methylnicotinamide and pyridoxal oxidizing activities in male mice increase in a parallel fashion upon the attainment of sexual maturity. (5) Competition of N1-methylnicotinamide and pyridoxal for oxidation in vivo has been demonstrated in the rat. (6) The inactivation of N1-methylnicotinamide oxidase activity which is connected with catalytic performance results in an equal loss of pyridoxal oxidase activity. A parallel loss in activity for both substrates also results from the catalysis of pyridoxal oxidation. In the course of these studies, a genetic analysis of aldehyde oxidase in the C58/J and C57B1/6J strains of mice was conducted. The earlier conclusion that aldehyde oxidase is controlled by a single autosomal locus with alleles acting in a codominant manner has been confirmed.

The biosynthesis of chelidonic acid (γ-pyrone-2,6-dicarboxylic acid) has been studied in mature, flowering Convallaria majalis L. plants. Glueose-U.L. −14 C and ribose-l −14 C were incorporated into the chelidonic acid molecule with dilutions of 720 and 940, respectively, while the dilution for acetate-2 −14 C was 11,000. Shikimate-G −14 C was not incorporated. Chemical degradation of chelidonic acid obtained from feeding glucose-6 −14 C showed 75% of the label in carbons 3, 4, and 5 and 25% in carbons 1, 2, 6, and 7. These results would preclude formation of chelidonic acid from sedoheptulose as a major pathway.

An acetylated dipeptide composed of equimolar quantities of aspartic acid and glutamic acid has been isolated in pure form from human brain by a combination of ion exchange and paper chromatographic techniques. Hydrazinolysis and acid hydrolysis studies indicate that the peptide is N -acetyl aspartyl glutamate. Preliminary synthesis studies suggest that the peptide may be of the alpha rather than the beta structural configuration.

Extensive pronase digestion of the desialized bovine submaxillary major mucin (BSM) resulted in a mixture of glycopeptides. Separation of the glycopeptides into nine fractions, fractions a to i , was achieved by linear gradient elution at constant pH (2.6) of 0.05–0.2 m pyridine-formate buffer from Dowex 50-X2 (pyridinium form). Glycopeptide b 6 was isolated from fraction b by preparative high-voltage paper electrophoresis, and glycopeptides g 5 and e 7 from fractions g and e , respectively, by preparative high-voltage paper electrophoresis and preparative paper chromatography, in succession. Homogeneity of these glycopeptides was shown by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of the N-terminal analysis by dansylation method with or without sequential Edman degradation and the analytical data before and after a β-elimination reaction of glycopeptides b 6 , g 5 , and e 7 indicated the structures of these glycopeptides as follows: Glycopeptide b 6 , seryl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide g 5 , glycyl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide e 7 , seryl-( O -α- N -acetylgalactosaminyl-)-threonyl-glycyl-( O -α- N -acetylgalactosaminyl-)-serine.

The addition of luteinizing hormone (LH) to preovulatory rabbit ovaries incubated in vitro stimulated steroid synthesis. When protein synthesis was inhibited by cycloheximide or puromycin, the effect of LH on Steroidogenesis was also blocked. Actinomycin D, at concentrations which limit incorporation of cytidine-H 3 into RNA to less than 10% of that of controls, did not affect steroid synthesis. Translational control or regulation of protein synthesis at a step following synthesis of messenger RNA may regulate steroid synthesis in this system.

A peptide composed of equimolar quantities of glutamic and aspartic acids was isolated from a protein-free extract of human brain by a combination of ion-exchange and paper chromatography. Hydrazinolysis studies established that glutamic acid was C-terminal and that the amino group of the aspartyl residue was substituted with an acetyl group. To determine which aspartyl carboxyl group was linked to glutamic acid, N -acetyl-α- and N -acetyl-β-aspartyl glutamate were synthesized. When the partial acid hydrolyzates of the synthesized acetylated peptides were chromatographed on the amino acid analyzer, two distinct elution patterns were observed. The elution pattern of the brain dipeptide resembled the alpha rather than the beta derivative. When acetylated amino acids prepared from brain dipeptide were incubated with hog l -specific kidney acylase, aspartic and glutamic acids were liberated.

Aerobacter aerogenes was found capable of growing well in the medium containing S -(β-aminoethyl)- l -cysteine, a metabolic antagonist of l -lysine, as a sole nitrogen source. The metabolic product has been isolated, purified, and identified as S -(β- N -acetyl-aminoethyl)- l -cysteine by comparison with the synthetic compound on the basis of studies by paper electrophoresis, paper and ion-exchange chromatographies, nuclear magnetic resonance spectroscopy, infrared spectrophotometry, X-ray diffraction, absorption spectrophotometry of ninhydrin color, elemental analysis, optical rotation, and reaction with l -lysine decarboxylase. The metabolic function of this N -acetylation is discussed.

1. 1. Methods were developed for the separation of small quantities (order of 10–50 μg.) of saturated higher fatty acids (C 12 –C 24 ). Filter paper coated with paraffin oil or latex served as a supporting medium in reversed-phase chromatography. 2. 2. Procedures were worked out for the demonstration of the fatty acid spots as lead sulfide or lead rhodizonate. Recognition of the fatty acids by bromothymol blue was found to be somewhat less sensitive.

The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit KmS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase.

Mixtures of ketones and acetic acid, namely 2-pentanone-acetic acid 30:2, 3-methyl-2-butanone-acetic acid 30:3, 4-methyl-2-pentanone-acetic acid 30:2, and 2, 6-dimethyl-4-heptanone-acetic acid 30:7 were found to give excellent separations of individual phospholipides on paper. It was possible to separate lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lecithin, and phosphatidic acid in a unidimensional chromatogram. The individual phospholipides had to be present at concentrations from 10 to 25 /μg./20 μl. in order to achieve good separations.

The conditions for the quantitative determination of melibiose (or raffinose after invertase hydrolysis) on paper chromatograms by either the use of “Genochrome” or N,N -diethyl- p -phenylenediamine as dip reagents were determined. The maximum density of the spots produced by the reaction of the chromogenic reagent with the sugar was measured by reflected light of 515 mμ wavelength. A linear relationship between the logarithm of the sugar concentration and the reflection density reading was observed with sugar concentrations between 20 and 75μg./5 μl. and, therefore, was the most favorable range in which to make the measurements. When the samples and the sugar standards are measured on the same paper chromatogram, the replicates agree within ±5%; between several chromatograms prepared the same day, replication values will be within ±10%.

An enzyme, oligogalacturonide trans-eliminase (OGTE), which degrades O-(4-deoxy-β- l -5-threo-hexopyranos-4-enyluronic acid)-(1 → 4)- d -galacturonic acid (“unsaturated digalacturonic acid”) into two molecules of 4-deoxy-5-hexoseulose uronic acid, has been purified from cell-free extracts of Erwinia carotovora. This enzyme also cleaves normal digalacturonic acid to one molecule of d -galacturonic acid and one molecule of the deoxyketuronic acid, indicating an eliminase (“lyase”) reaction. Polygalacturonic acid is attacked by purified OGTE at 1 400 the rate of unsaturated digalacturonic acid. The rate of attack by OGTE on oligogalacturonides varies inversely with the chain length of the substrate. The monomer, galacturonic acid, is not converted to the deoxyketuronic acid by OGTE. Erwinia OGTE has a pH optimun of approximately 7.2, and it does not require calcium ions for maximum activity. In the presence of Erwinia cell-free extracts and NADH, the deoxyketuronic acid is converted to 2-keto-3-deoxy- d -gluconic acid, presumably with the intermediate formation of 3-deoxy- d -glycero-2,5-hexodiulosonic acid. Two pathways are thus involved in the metabolism of pectic substances by Erwinia carotovora: one route through galacturonic acid (involving galacturonic acid isomerase), and the other through unsaturated digalacturonic acid (involving OGTE) with the formation of 4-deoxy-5-hexoseulose uronic acid. Both pathways yield 2-keto-3-deoxy- d -gluconic acid as a common product.

Application of four independent methods for cystine indicate that there are eight half-cystines per mole of Escherichia coli B asparaginase, consistent with a four subunit model with one intrachain disulfide link per subunit. Treatment with dinitrofluorobenzene showed only leucine as NH 2 -terminal amino acid; values of 3.17–3.9 moles of DNP-Leu per mole of asparaginase were obtained. Hydrazinolysis released tyrosine as the principal amino acid, 3.3 residues per mole of protein being obtained. Studies with the carboxyl modification technique of Hoare and Koshland ( Hoare, D. G., and Koshland, D. E. (1967) J. Biol. Chem. 242, 2447 ) showed that 61% of the side chain carboxyl groups are present as amide. Studies with carboxypeptidase A demonstrated the carboxyl terminal sequence to be -(Ile, Glu, Asn)-Phe-Ile-Gln-Tyr. The results obtained by preparing two-dimensional peptide maps of the tryptic digest of reduced, S -aminoethylated enzyme are also in agreement with a four subunit model in which the subunits are identical or nearly so.

Action of pullulanase from Aerobacter aerogenes has been tested on a number of starch oligosaccharides. Hydrolysis is confined to cleavage of α-1 → 6-interchain links between oligosaccharide chains containing a minimum of two glucose units per chain. Starch oligosaccharides and dextrins containing two or more α-1 α 6-links are also cleaved. Glucose has not been observed as a product. A single α-1 α 6-linked glucose “stub” does not interfere with pullulanase action, and oligosaccharides containing such stubs are found in the products of pullulanase action. Under special conditions, pullulanase preparations exhibit condensation and transferase reactions.

The effect of the female hormones on glycoproteins and glycosaminoglycans in uteri has been studied. The uteri taken from the ovariectomized rabbits treated with estrogen, estrogen plus progesterone, and sham administration (control) were incubated in vitro with [U- 14 C]glucose. Subsequently, the tissues were digested extensively with pronase, yielding crude glycan fractions. The amount and radioactivity of the crude glycan fraction increased by the treatment with estrogen, but reduced to certain level with progesterone. Separation of glycoproteins and glycosaminoglycans was achieved by stepwise elution from Dowex 1 (X2, chloride form) with increasing concentration of NaCl. The yield and radioactivity, together with the results of chemical, enzymatic, and electrophoretic studies on the resulting fractions indicated that the metabolism of a slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulf ated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulf ate were stimulated remarkably with estrogen, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was, however, found to be different from each other. It was noticed that sulfated glycoproteins were the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of a neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone.

The copper-catalyzed alkaline oxidation of thiomalic acid (TMA) to the corresponding disulfide occurs with the metal firmly bound in a stable TMA-Cu II complex, which is characterized by a sharp absorption peak with maximum at 340 nm. In this complex the TMA to copper ratio is 2:1. The complex remains unchanged during TMA oxidation by molecular oxygen, and quickly disappears at the end of the reaction. In the absence of oxygen the TMA-Cu II complex slowly disappears, Cu II being reduced by the excess of TMA. In the course of TMA oxidation an intermediate compound, showing a characteristic absorption band centered at 300 nm, accumulates in the solution, and quickly disappears at the end of the reaction, in coincidence with the complete oxidation of TMA. It has been demonstrated that the amount of the 300-nm absorbing compound is proportional to the initial TMA concentration and to the oxygen concentration, while is independent of the concentration of TMA-Cu II complex. It may possibly represent an intermediate between TMA and the disulfide in which oxygen is involved. H 2 O 2 is produced during TMA oxidation, accumulates in the reaction mixture in amounts proportional to the oxygen concentration, and is quickly destroyed at the end of the reaction, when Cu II is set free from the TMA-Cu II complex.

Tritiated cyanocobalamin ([3H]cyano-B12) uptake was maximal at pH 6.0–8.0, required phosphate buffer, was proportional to the cell concentration, was inhibited reversibly by Tris+, and was markedly temperature dependent (optimal, 37–47 °). Starved cells showed a 3–4-fold dependency on glucose and their glucose-dependent uptake was decreased by certain inhibitors of energy metabolism. However, the primary role of glucose in uptake may be to maintain cell wall structure rather than to supply chemical energy for transport per se. The Km for [3H]cyano-B12 uptake was 1.0 × 10−8 m and the Vmax was 2.3 pmoles/min/mg of dry cell wt. Unchanged [3H]-cyano-B12 is the major cobalamin after 5 min of uptake. Treatment with Tris-EDTA reduced the initial uptake rate by 40–50%, but osmotic shock decreased it an additional 5–8-fold. No detectable 5-methyltetrahydrofolate-homocysteine B12 methyltransferase was released by osmotic shock. However, treatment with Tris-EDTA alone, as well as osmotic shock, did release from the cells a heat-labile, ammonium sulfate-precipitable, binding protein. By gel filtration, its molecular weight and Stokes radius were 22,000 and 15.6 A, respectively. A very large binder (mol wt >200,000) was also found in Tris-EDTA and shock fluids.The 4 ° dissociation constant for the reversible complexes between [3H]cyano-B12 and both binders was 0.6 × 10−8 m . At 31 ° it was 0.5 × 10−8 m .