Herman M. Kalckar

The Periplasmic Galactose Binding Protein of Escherichia coli

A specific high affinity galactose transport system called Pβg can be induced by trace amounts of galactose in the medium by virtue of its own ability to capture and accumulate galactose. The transport system is coregulated with the production of a high affinity periplasmic galactose binding protein, which constitutes but one part of the transport system. Some transport negative mutants still remain producers of this binding protein. A close correlation exists between production of the active binding protein and the presence of galactose chemotaxis. The hypothesis, that this binding protein is a common element of the specific galactose transport system, Pβg, and of galactose chemotaxis is supported by observations on structural mutants, being defective in galactose binding protein as well as showing a lack of galactose chemotaxis. The binding protein is a monomer with two binding sites for galactose. Binding of one or two of the galactose molecules elicits specific conformational changes of the galactose binding protein (lowered affinity for galactose, increase of charges of the protein, increased fluorescence of tryptophan residues). The importance of these features for transport and for chemotaxis is discussed (70).

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Abstract Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar

2-Amino-4-hydroxy-6-formylpteridine, an inhibitor of purine and pterine oxidases☆

Abstract Most folic preparations are found to exert an inhibitory effect on xanthopterin and xanthine oxidase. This seems to be due to the presence of an impurity in the preparations. The inhibitor is supposed to be 2-amino-4-hydroxy-6-formylpteridine. The inhibitory activity is destroyed by irradiation with light, reduction with metallic zinc or by binding of the aldehyde group with 2,4-dinitrophenylhydrazine. A colorimetric method for the estimation of the aldehyde is given. By incubation of the aldehyde with xanthine oxidase the inhibitory activity almost completely disappers. The enzymatic conversion of the aldehyde can be followed both bythe changes in the fluorescence and in the absorption spectrum of the compound.

[20] Enzymes of the Leloir pathway

Publisher Summary This chapter focuses on the enzymes of the leloir pathway, and describes the quantitative determination of enzymatic activity in broken cell preparations. Specific enzymatic assays designed especially for use with crude bacterial extracts are described. Galactokinase catalyzes the reaction ATP + Gal→ ADP +α -Gal-l-P. The Gal-1-P produced in a preincubation mixture is quantitatively determined. The Gal-l-P formed is determined in the proteinfree filtrate by an analytical incubation mixture. The absolute molar amount of TPNH formed is a direct measure of Gal-l-P and is thus a measure of galacto-kinase activity. The rate of G-1-P formation in Gal-l-P uridyl transferase is analyzed by adding the two indicator enzymes and coenzymes in excess, phosphoglueomutase (plus glucose-l,6-diphosphate) and G-6-P dehydrogenase plus TPN. In UDPGal-4-epimerase, UDPG formed is determined in a protein-free filtrate by means of a DPN-dependent specific dehydrogenase (UDPG dehydrogenase). The chapter also describes the purification of galactose-l-phosphate uridyl transferase, and purification of UDPgalactose-4-epimerase.

The role of phosphoglycosyl compounds in the biosynthesis of nucleosides and nucleotides.

Abstract In the biosynthesis of nucleosides and nucleotides the intermediate formation of ribose (or deoxyribose)-1-phosphate and their corresponding 5-esters may play a prominent role in the animal organism; the pathway seems to involve a phosphorylation (by ATP) of the 1 position of ribose-5-phosphate yielding ribose-1,5-diphosphate. This new diester can also be obtained from ribose-1-phosphate and glucose-1,6-diphosphate in the presence of phosphoglucomutase. In pigeon liver extracts there are strong indications that ribose-1,5-diphosphate can exchange its 1-ester phosphate with adenine, hypoxanthine or incomplete purine precursors. Glucose-1-phosphate and galactose-1-phosphate (both α-glycosyl compounds) also play a role as acceptors of uridyl radicals catalyzed by a special class of enzymes, uridyl transferases. Uridine triphosphate plus one of these enzymes incubated with one or the other of the two above-mentioned 1-esters yields Leloirs UDP-glucose or UDP-galactose, the so-called “CoWaldenases”. Conversely, the latter compound incubated with inorganic pyrophosphate and a uridyltransferase gives rise to the formation of uridine triphosphate (UTP). UTP may, through the action of another uridyl transferase, play a role in the incorporation of the uridyl radical into nucleic acids.

Fluorescence enhancement of uridine diphosphogalactose 4-epimerase induced by specific sugars

Abstract It has been found that 5′-uridylic acid (5′-UMP) and some specific sugars, related to induction and repression of the biosynthesis of UDPGal-4-epimerase, exerted a concerted action on UDPGal-4-epimerase fluorescence. This is manifested by a severalfold increase of blue fluorescence due to bound DPNH of epimerase. Among the uridine nucleotides, 5′-UMP (at 10−4 m ) is highly active. Other 5′-mono- or diphosphonucleosides were much less active. Synthetic UDP-glucose and UDP-galactose were inactive. The specific sugars (10−2–10−3 m ) which show strong activity are d -fucose > d -galactose > d -glucose > d -xylose, and l -arabinose; l -fucose and sucrose are inactive.

Role of the galactose transport system in the retention of intracellular galactose in Escherichia coli.

Abstract A galactose transport system with a high affinity for galactose, Rotmans (β-methyl galactoside permease, has been shown to be essential for the internal induction of the galactose operon in galactokinaseless mutants of Escherichia coli . As the result of a second mutation affecting the β-methylgalactoside permease gene, galactokinaseless mutants were found to have lost the internal induction of the galactose operon. Evidence has been previously presented indicating that this double mutant fails to retain galactose which is endogenously generated from uridine diphosphate galactose. Such a failure in the retention mechanism allows the endogenously generated galactose to be lost into the medium and the intracellular level of galactose to drop below the threshold for the induction of the galactose operon. Comparison of the rates of entry and exit of galactose in this double mutant with the rates in the parental strain has revealed that the mutant loses its internally generated galactose, not by an increase in the rate of exit, but by a marked decrease in the rate of entry. These observations are consistent with the view that the recapture of galactose by a highly efficient permease is responsible for the retention of intracellular galactose.

The metabolic basis for masking of receptor-sites on E. coli K-12 for C21, a lipopolysaccharide core-specific phage

Abstract Further studies on the capacity of the cell envelopes of Escherichia coli K-12 to adsorb a bacteriophage C21 led to the following observations. Epimerase-defective mutants adsorb C21; this applies to a mutant with a slightly leaky epimerase defect as well as to one with no trace of epimerase. Addition to the growth medium of galactose (8 × 10 −6 m ) for 1–2 generations markedly decreased and in some cases practically eliminated the capacity of these mutants to adsorb C21. The small amounts of galactose which contribute to the effective masking of the receptor sites seem to be incorporated as galactosyl units in the lipopolysaccharide (LPS). Purified LPS preparations from the epimeraseless mutants were able to inactivate C21. The LPS from the mutants with a complete block of epimerase were particularly effective in adsorbing C21; this LPS contained only minute traces of galactose. The LPS from the leaky epimerase mutants contained considerable amounts of galactose, but could nevertheless adsorb C21 quite effectively. LPS from UDPG defectives, which is practically devoid of glucose as well as galactose, has no detectable adsorption capacity. LPS from cells with a normal UDPGal (such as wild type, K − or T − strains) had practically no capacity to adsorb C21. However, boiling of these preparations at pH 5.5 apparently unmasked C21 receptor sites. The most conspicuous chemical change after the boiling was the release of all the 2-keto-3-deoxy-octulosonate (KDO) from the LPS as free KDO. This type of removal of KDO from LPS had no effect on the inert LPS from the UDPG defectives. It seems, therefore, that a certain fraction of LPS galactose and quite possibly a part or all of the KDO contributes to the masking of C21 receptor sites in wild type K-12.

The nature of regulation of hexose transport in cultured mammalian fibroblasts: Aerobic “repressive” control by d-glucosamine

Abstract Restrictive control (“repression”) of 3- O -methylglucose transport (or of galactose uptake) in confluent NIL hamster fibroblast cultures was found to be highly pronounced after preconditioning the cultures in medium containing d -glucosamine. The “repression” exerted by glucosamine developed slowly over several hours. The transport “repression” was counteracted by anaerobiosis, by 2,4-dinitrophenol ( H. M. Kalckar, C. W. Christopher, and D. Ullrey, 1979 , Proc. Nat. Acad. Sci. USA 76 , 6453–6455), and by fluoride as well as by malonate. In “de-repressed” cultures, i.e., in the absence of glucosamine in the medium or by using fructose during preconditioning, malonate did not affect regulation of the hexose transport system. In culture medium deprived of l -glutamine and serum, repressive control of the transport system by glucose as well as by glucosamine was greatly aggravated. However, the simultaneous addition of malonate abolished the severe “repression” by either of the hexoses. In all cases, preconditioning with fructose permitted high (“de-repressed”) transport activity. Unlike glucose, galactose, or glucosamine, fructose was not found to compete in the transport assay. The metabolic inhibitors which prevent the aerobic curtailment of the hexose transport system are all more or less directly interfering with the flow of metabolites through the tricarboxylate cycle, which may therefore play an important role in the “repressive” control of transport.

The biological incorporation of purines and pyrimidines into nucleosides and nucleic acid.

Abstract The mechanism of incorporation of purines and pyrimidines into ribosidic linkage has been discussed from various points of view. Results gained from enzymatic studies are not in direct agreement with observations made in intact organism using isotopes. Various ways of interpretations are discussed.