Gene-Enzyme Relations of Tryptophan Mutants in STREPTOMYCES COELICOLOR A3(2)

Mutations in twenty-eight tryptophan mutants of S. coelicolor A3(2) were mapped relative to the nearest flanking markers. Mutants lacking single enzymatic activities for phosphoribosyltransferase, phosphoribosylanthranilate isomerase, indodeglycerol phosphate synthase, tryptophan synthase A and tryptophan synthase B were identified.     

Dry weight accumulation and starch-synthesis enzymes in grain of cultured ears of three winter wheat varieties

Detached ears of three winter wheat ( Triticum aestivum L.) varieties were cultured in solution for 12 days with sucrose levels varying from 36.5 to 292 m M. The dry weight and starch content of grains increased asymptotically with the sucrose level in the solution. At 4 days of culture, glucose phosphate isomerase (EC 5.3.1.9) activity grain⁻¹ was lower with 36.5 m M than with higher sucrose levels in the medium; at 8 days, adenosinc diphosphoglucose pyrophosphorylase (EC 2.7.7.27) and (soluble plus bound) starch synthase (EC 2.4.1.21) activities grain⁻¹ were higher with 146 and 292 m M sucrose than with 36.5 and 73 m M sucrose. The multiple regression of starch content over these enzyme activities showed that starch synthase was relatively more important as an independent variable. The dry weight and starch content of grains were higher in the variety Maris Huntsman than in Splendeur and Hobbit. The water content of grains was lower in Splendeur than in the other two varieties. At 4 days the glucose phosphate isomerase, adenosine diphosphoglucose pyrophosphorylase and starch synthase activities grain⁻¹ were smaller in Splendeur than in Hobbit and Maris Huntsman and al 8 days they were higher in Maris Huntsman than in Hobbit and Splendeur. The varietal differences in starch content of grains were related to the activities of glucose phosphate isomerase and especially of starch synthase.     

Partial Characterization of Phosphoribosyl Transferase, Phosphoribosyl Anthranilate Isomerase, and Indole Glycerol Phosphate Synthase from Serratia marcescens

The molecular organization of the enzymes phosphoribosyl (PR) transferase, phosphoribosyl anthranilate (PRA) isomerase, and indole glycerol phosphate (InGP) synthase of the tryptophan biosynthetic pathway of Serratia marcescens was investigated and compared with that reported in other enteric bacteria. PRA isomerase and InGP synthase activities were found to reside in a single polypeptide chain, a situation analogous to that in Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes. This bifunctional enzyme was purified to near homogeneity. Its molecular weight was estimated to be 48,000. PR transferase was found unassociated with PRA isomerase and InGP synthase after gel filtration and ion-exchange chromatography. Whereas in other enteric organisms PR transferase has been reported to form an aggregate with anthranilate synthase, it is a distinct entity in S. marcescens.     

Organization of the functional domains of anthranilate synthase from Neurospora crassa. Limited proteolysis studies

Treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of Neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (InGP) synthase and N-(5'-phosphoribosyl)anthranilate (PRA) isomerase activities. Sequencing the NH2 terminus of the InGP synthase-PRA isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment of the polypeptide chain which links the glutamine-binding (G) domain with the InGP synthase-PRA isomerase domains. The fragment containing anthranilate synthase activity has a molecular weight of 98,000, as estimated by gel filtration, and is composed of an apparently intact alpha-subunit (70 kDa) associated with the G-domain fragment (29 kDa) derived from the beta-subunit. The alpha X G-domain complex was resistant to further degradation by elastase. When either the alpha 2 beta 2 complex or the alpha X G-domain complex was incubated with trypsin, the alpha-subunit was degraded to a 66-kDa alpha-fragment with reduced enzymatic activity, which was resistant to further cleavage. In contrast, incubation of alpha-subunit alone with either elastase or trypsin resulted in its complete degradation, indicating that association of the alpha-subunit with either G-domain or beta-subunit protected the alpha-subunit from this extensive degradation. A model for the anthranilate synthase complex is proposed in which the trifunctional beta-subunit forms a dimer by the self-association of the InGP synthase-PRA isomerase domains; the G-domain is connected to the InGP synthase-PRA isomerase domain by a relatively disordered region of the peptide chain which, in the alpha 2 beta 2 complex, remains susceptible to proteases; and neither alpha-subunit nor G-domain significantly self-associates.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Inhibition of pulmonary thromboxane A2 synthase activity and airway responses by CGS 13080

The effects of CGS 13080, a thromboxane (TXA₂) synthase inhibitor, on airway responses to arachidonic acid (AA) were investigated in the anesthetized cat. Feline and human lung microsomal fraction exhibited prostaglandin I₂ (PGI₂, prostacyclin), and TXA₂ synthase activities, and human platelet microsomal fractions exhibited TXA₂ synthase activity. Cat and human lung microsomal fractions, but not human platelets, exhibited the presence of GSH-dependent PGE₂ isomerase activity. CGS 13080 inhibited TXA₂ synthase activity in all three microsomal fractions in a concentration-dependent manner. The increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance in response to AA were decreased significantly by CGS 13080. These data suggest that the bronchoconstrictor actions of AA are mediated in large part by the formation of TXA₂. The data further indicate that cyclooxygenase products other than TXA₂ are involved in the bronchoconstrictor response to AA since meclofenamate had greater inhibitory activity than did CGS 13080. Moreover, the effects of CGS 13080 were due to inhibition of TXA₂ synthase rather than an effect on TXA₂ receptors, since airway responses to the TXA₂ mimic, U46619, were not altered. The present data show that CGS 13080 inhibits TXA₂ synthase activity without altering cyclooxygenase, PGI₂ synthase, or GSH-dependent PGE₂ isomerase activities. The data further indicate that in vivo administration of CGS 13080 may selectively increase PGI₂ synthase activity.     

Functional analysis of human P5, a protein disulfide isomerase homologue

Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

Cloning and sequencing analysis of Trp1 gene of Flammulina velutipes

The genomic TRP1 gene from basidiomycete Flammulina velutipes was cloned by complementation of yeast Saccharomyces cerevisiae trp1 mutation. Sequencing analysis revealed that the TRP1 gene encoded a single protein consisting of three catalytic functional domains; glutamine amidotransferase, indole-3-glycerol phosphate synthase ) and N-(5'-phosphoribosyl) anthranilate isomerase, in order of NH2-glutamine amidotransferase-indole-3-glycerol phosphate synthase N-(5'-phosphoribosyl) anthranilate isomerase-COOH. The coding sequence of the TRP1 gene was interrupted by a single intron of 48 bases, the position and flanking sequences of which were highly homologous to those of basidiomycete Phanerochaete chrysosporium trpC.     

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.     

Lipoprotein lipase, a key role in atherosclerosis?

Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well-suited for crystallisation attempts. Two enzymes of histidine biosynthesis from Thermotoga maritima, N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed in Escherichia coli, both in their native and seleno-methionine-labelled forms, purified by heat precipitation of host proteins and crystallised. N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X-ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution. The obtained crystals will enable the determination of the first three-dimensional structures of enzymes from the histidine biosynthetic pathway.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes

Splenic lymphocytes from mice immunized with a partially purified prostaglandin (PG) H-PGE isomerase from sheep vesicular glands were fused with SP2/0-Ag14 myeloma cells. Two spleen cell-myeloma hybrids (hei-7 and hei-26) were selected and cloned. The mouse antibodies secreted by the two hybrids, IgG1 (hei-7) and IgG1 (hei-26), caused immunoprecipitation of a maximum of 45 and 22%, respectively, of the solubilized PGH-PGE isomerase activity of sheep vesicular gland; immunoprecipitation of activity by the two antibodies was additive. The antigens reactive with IgG1 (hei-7) and IgG1 (hei-26) were identified as proteins with Mr = 17,500 and 180,000, respectively, by Western transfer blotting or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated 125I-labeled microsomes. The PGH-PGE isomerase activities precipitated by IgG1 (hei-7) and IgG1 (hei-26) exhibited different kinetic properties with respect to time course, Km for PGH2, and concentration dependence for GSH. No significant GSH-S-transferase activity was present in these immunoprecipitates. These data indicate that there are at least two different proteins in sheep vesicular gland microsomes capable of catalyzing GSH-dependent PGH-PGE isomerase reactions. IgG1 (hei-7), but not IgG1 (hei-26), caused coprecipitation of PGH synthase and PGH-PGE isomerase activities when incubated with intact right-side-out vesicular gland microsomes. Thus, the epitope for IgG1 (hei-7) is located on the cytoplasmic surface of those microsomal spheres which contain PGH synthase. This latter finding suggests that the isomerase reactive with IgG1 (hei-7) is involved in PGE synthesis in sheep vesicular glands.     

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.     

Glycolytic enzymes in mammalian spermatozoa. Activities and stabilities of hexokinase and phosphofructokinase in various fractions from sperm homogenates

1. Methods of homogenizing suspensions of washed mammalian spermatozoa were studied. The most useful methods were those using sonication and those using a French press. 2. Hexokinase, phosphofructokinase, glucose phosphate isomerase and adenosine triphosphatase activities in ram, bull and boar spermatozoa were investigated by using these two homogenization methods. Glucose phosphate isomerase, representative of soluble cytoplasmic material, was very readily extracted and remained entirely in the supernatant after centrifugation at 145000g for 60min. In contrast, the other three activities were less easily extracted and were sedimented in various proportions under the described conditions of centrifugation. 3. Attempts to obtain subcellular fractions from sperm homogenates by ;classical' methods failed, owing apparently to the inhomogeneity of subcellular particles in the homogenates. It is concluded that, after removal of sperm heads, the only meaningful fractionation is a separation of spermatozoal material which sediments at 145000g during 60min from that which does not. 4. The stabilities of hexokinase and phosphofructokinase activities in bull, boar and ram sperm homogenates were investigated. Hexokinases showed very little dependence on the various environments tested, whereas the optimum conditions for phosphofructokinase stability were: a minimum of sonication, the presence of phosphate ions and of a thiol-group protectant, and a pH7.5. Activities of hexokinase, phosphofructokinase and glucose phosphate isomerase per sperm cell were compared with published data on rates of fructolysis by spermatozoa; the potential catalytic activities were shown to be considerably in excess of these rates. However, phosphofructokinase may be the rate-limiting enzyme of glycolysis in vivo in bull and ram spermatozoa.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Tryptophan Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.