Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, α-D-galactose-1-phosphate:UDP glucose uridylyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increased cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.     

基因工程改良淀粉品质

淀粉对人类生活十分重要,它不仅是人们的能量和营养来源,而且还是重要的工业原材料。对于淀粉合成过程及淀粉的加工、使用一直是淀粉研究的重点内容。淀粉的合成在最后阶段涉及到３个关键性的酶是：ＡＤＰＧ焦磷酸化酶、淀粉合成酸以及淀粉分支酶。它们分别催化ＡＤＰ－葡萄糖的形成、葡聚糖链的延伸以及分支链的形成。另外淀粉去分支酶对淀粉最终结构的形成也起到重要作用。本文将介绍上述４个酶近年来的生物化学和分子生物学研究     

Regulation of the purine catabolic enzymes in Neurospora crassa.

Neurospora crassa can utilize purines and their metabolic products as a nitrogen source. Regulation of the five enzymes required for uric acid metabolism was studied. The first three enzymes of this catabolic pathway are controlled in a complex manner that involves both induction and repression. Both uricase and allantoicase were induced by uric acid while allantoinase was induced by either uric acid or allantoin. Synthesis of all three of these enzymes was repressed by the end product, ammonia. The ure-2 mutant, which is urease deficient and cannot derive ammonia from purines, shows a hyperinducibility of these same three enzymes. The last two enzymes of the pathway, ureidoglycollase and urease, were found to be constitutive.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases.

UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase was partially purified from rat liver Golgi membranes and rat serum. The kinetic parameters of the two enzymes isolated by affinity chromatography were compared with each other and with those for commercial bovine milk galactosyltransferase. When N-acetyl-glucosamine was the acceptor the Km values for UDP-galactose were 65,52 and 43 microM for the rat liver Golgi, rat serum and bovine milk enzymes respectively. The Km values for N-acetylglucosamine were 0.33, 1.49 and 0.5 mM for the three enzymes respectively. The Km values for UDP-galactose, with glucose as acceptor in the presence of 1 mg of alpha-lactalbumin, were 23, 9.0 and 60 microM for the three enzymes respectively, and the Km values for glucose were 2.3, 1.8 and 2.0 mM respectively. The effects of alpha-lactalbumin in both the lactosamine synthetase and lactose synthetase reactions were similar. The activation energies were 94.0 kJ/mol (22.5 kcal/mol) and 96.0 kJ/mol (22.9 kcal/mol) for the Golgi and serum enzymes respectively. Although some differences in Km values were observed between the rat liver Golgi and serum enzymes, the values obtained suggest a high degree of similarity between the kinetic properties of the three galactosyltransferases.     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

The Apparent Conservation of the Internal Low Efficiency Promoter of the Tryptophan Operons of Several Species of ENTEROBACTERIACEAE

The specific activities of the tryptophan biosynthetic enzymes were determined for eleven different species of Enterobacteriaceae grown under repressing and non-repressing conditions. In all the bacteria examined the multipliciy of derepression for the first two enzymes of the pathway was at least twofold greater than the multiplicity of derepression for the last three enzymes.     

Determination of some biochemical and structural features of alcohol dehydrogenases from Drosophila simulans and Drosophila virilis. Comparison of their properties with the Drosophila melanogaster Adhs enzyme.

The biochemical properties of the enzyme alcohol dehydrogenase of two different Drosophila species, Drosophila simulans and Drosophila virilis, were studied and compared with those of Drosophila melanogaster Adhs enzyme. All of them consist of two identical subunits of molecular weight 27800 and share significant similarities in function. The substrate specificities of these enzymes were characterized and Km(app.) and Vmax.(app.) values were calculated. All these alcohol dehydrogenases show greater affinity for secondary rather than for primary alcohols. The amino acid compositions of the three enzymes were determined, and there is a close similarity between the D. simulans and the D. melanogaster enzymes, but there are significant differences from the alcohol dehydrogenase of D. virilis. The N-terminal amino acid is blocked and the C-terminal amino acid is the same for all three alcohol dehydrogenases. The enzymes from the three species were carboxymethylated and digested with trypsin. The peptide 'maps' reveal, as expected, more homologies between the enzymes of D. simulans and D. melanogaster than with the enzyme of D. virilis.     

Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.     

Extended stability of restriction enzymes at ambient temperatures

The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.     

Functional activity of the mouse flavin-containing monooxygenase forms 1, 3, and 5

Three functional mouse flavin-containing monooxygenases (mFMOs) (i.e., mFMO1, mFMO3, and mFMO5) have been reported to be the major FMOs present in mouse liver. To examine the biochemical features of these enzymes, recombinant enzymes were expressed as maltose-binding protein fusion proteins (i.e., MBP-mFMO1, MBP-mFMO3, and MBP-mFMO5) in Escherichia coli and isolated and purified with affinity chromatography. The substrate specificity of these three mouse hepatic FMO enzymes were examined using a variety of substrates, including mercaptoimidazole, trimethylamine, S-methyl esonarimod, and an analog thereof, and a series of 10-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazine analogs. The kinetic parameters of the three mouse FMOs for these substrates were compared in an attempt to explore substrate structure--function relationships specific for each mFMO. Utilizing a common phenothiazine substrate for all three enzymes, we compared the pH dependence for the recombinant enzymes under similar conditions. In addition, thermal stability for mFMO1, mFMO3, and mFMO5 enzymes was examined in the presence and absence of NADPH. The results revealed unique features for mFMO5, suggesting possible impact on the functional significance of this abundantly expressed FMO5 isoform in both human and mouse liver.     

海藻糖对医用诊断工具酶活性保护研究

研究了干燥条件下海藻糖对胆固醇氧化酶,胆固醇脂酶和过氧化物酶的活性保护作用,并对影响酶活性保存的其它因素进行了试验。加海藻糖干燥的以上三种诊断酶分别在70,55和37℃温度下放置40d,150d和210d后再次测定酶活,结果表明这三种酶的活性均保持在90％以上,而未加海藻糖保护的对照,活性降至20％以下。除海藻糖浓度外,缓冲系统中的介质、离于强度、pH是影响酶活的关键因素。     

白腐菌木质素降解酶及其在木质素降解过程中的相互作用

木质素是一类不易降解的生物物质,在自然界中,白腐真菌对木质素的降解能力最强.白腐真菌降解木质素主要依靠分泌的三种酶:木质素过氧化物酶(Lip)、锰过氧化物酶(MnP)和漆酶(Lac).对白腐真菌分泌的三种木质素降解酶在性质、分布等方面进行了比较,系境地介绍三种木质素降解酶的催化作用,并阐述其在木质素降解过程中的相互作用.     

Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated.

The enzymes for galactose metabolism in Saccharomyces cerevisiae are encoded by three tightly linked genes. Data presented in this paper show that, in contrast to enzymes encoded by other gene clusters in yeast, these three enzymes are translated as separate polypeptides. First, two of the enzymes encoded by the cluster, galactokinase and uridylyl transferase. purified to near homogeneity, are separate polypeptides. Second, no precursor polypeptide-containing sequences common to both these enzymes is detectable in extracts from galactose-induced yeast cells. Third, no partial or absolute polarity of expression of the enzymes is observed in strains containing nonsense mutations in any of the genes of the cluster.Expression of the three galactose metabolic enzymes is co-ordinate, both during induction and during steady-state synthesis. This is true both for wild-type yeast strains and for strains carrying the long-term galactose adaptation mutation, gal3. In GAL3⁺ strains mutations within the galactose gene cluster have no effect on this co-ordinate expression. However, in gal3^? strains, mutations in any of the genes of the cluster completely eliminate expression of the other two genes. These results suggest that the GAL3 gene product is responsible for inducer synthesis and that the actual inducer is an intermediate in galactose metabolism.     

Effects of high-carbohydrate diets on lipogenesis in rat interscapular brown adipose tissue

Cycloplasmic preparations from brown and white adipose tissues were assayed for three lipogenic enzymes throughout a programme of starvation followed by refeeding on either a normal or a white-bread diet. In the brown adipose tissue of rats fed on a white-bread diet the three enzymes were elevated to levels significantly higher than those in white adipose tissue.     

Regulation of leucine biosynthesis in Bacillus subtilis

The biosynthesis of alpha-isopropylmalate (alphaIPM) synthetase, IPM isomerase, and betaIPM dehydrogenase in Bacillus subtilis can be derepressed in leucine auxotrophs by limiting them for leucine. The derepression of the three enzymes is apparently coordinate. A class of mutants resistant to 4-azaleucine excretes leucine and has derepressed levels of all three enzymes. The azaleucine-resistance mutations may lie in a gene (azlA) encoding a repressor. Efforts to find mutations characteristic of a constitutive operator have been unsuccessful. No polar mutations have been found among nine leucine auxotrophs that have characteristics of frameshift mutations. The enzyme catalyzing the first step in leucine biosynthesis, alphaIPM synthetase, is sensitive to feedback inhibition by leucine. We conclude that leucine biosynthesis is controlled by the inhibition of the activity of the first biosynthetic enzyme by leucine, and by the repression of the synthesis of the first three biosynthetic enzymes by leucine. The repression of the three enzymes may be under the control of a single repressor and a single operator, or of a single repressor and a separate operator for each structural gene.     

Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall biosynthesis.

Within the past 18 months work has continued on the structure and mechanisms of enzymes involved in the diaminopimelic acid/lysine biosynthetic pathway. A novel structure has been determined for a PLP-independent epimerase, and structures with bound substrates have been solved for two other enzymes. Additionally, new studies have appeared describing the chemical mechanisms of three enzymes in the pathway.     

The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland.

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.