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Starch-granule bound glucan synthetase was isolated from cotton leaves by a sucrose density technique. Starch granules were successively washed with 0.02 M Na-phosphate buffer containing 0.9% NaCl. The alkaline hydrolysate of washed materials and acid hydrolysate of protein-bound amylopectin were separated by use of Con A-Sepharose. Amylolytic hydrolysate of starch granules also was carried out with α-amylase and fractionated by chromatography on Sephadex G-25. Amylopectin was the only glucan glycosidically linked with proteins, in which glucan synthetase activity was identified. The amylose component was free from covalently bound proteins.

A procedure to compare N fertilizers based on comparing complete response curves simultaneously represented by a quadratic multifertilizer response model (MRM) is discussed. A modified version of the MRM, called fertilizer testing model (FTM), is used to test differences among response curves. The FTM describes the response curve to a fertilizer selected as a standard as well as the additional effect of test fertilizers over the response to the standard. In case of different response curves, an approximate test to decide whether the curves have equal limiting yield is introduced. Finally, a procedure to estimate the availability coefficient from quadratic response curves having equal limiting yield is derived. These procedures were employed to analyze six field experiments conducted over a period of 3 years in Korea using three varieties of rice (Oryza sativa L. ‘Tongil,’ ‘Jinheung,’ and ‘Milyang 23’) and three fertilizers (urea and two sulfur-coated ureas, or SCU) applied at four or five levels of N. Two out of six experiments showed a significant effect from SCU-A (25% release/7 days) beyond that of split-applied urea, whereas SCU-B (14% release/7 days) showed significant additional effects in three out of five experiments. However, in four of these six experiments, the limiting yield of the response curves of SCU was significantly higher than that of urea, precluding a valid estimation of the availability coefficient. Possibly, the proper combination of N dose and timed release of N from SCU in these experiments translated into grain yields unmatched by split applications of urea.

α-Amylase activity from phenolic-rich plant tissues is successfully determined with an amylopectin-azure substrate. This procedure, due to the substrate's specificity for α-amylase, overcomes the effects of the interfering substances firmly associated with enzyme preparations. The procedure is more sensitive than the assay for activity with iodine solution.

A simple sucrose density technique has been developed for starch granule isolation from leaves. With this procedure starch-granule-bound glucan synthetase (EC 2.4.1.21) was successfully isolated from phenolic-rich plant tissue. This enzyme is firmly bound to starch granules and free from soluble glucan synthetase. The enzyme specific activity is more than 100-fold higher than that prepared by the previously reported procedure.

Glandless cotton plants (Gossypium hirsutum L. cv. Coker 100) were subjected to the influence of high CO(2)-bicarbonate. The content of protein decreased with no accompanying increase in its degraded products. The decrease in protein was correlated with the low content of chlorophyll and also with the reduced activity of carbonic anhydrase. The initiation of these correlations coincided with the time when the control leaves contained the highest enzyme activity during leaf growth. The high concentration of bicarbonate directly restricted the rate of photophosphorylation and that of the Hill reaction in isolated chloroplasts. The amount of ATP in leaves treated in vivo also diminished. High CO(2) as bicarbonate, however, did not directly inhibit the activity of carbonic anhydrase in vitro.

Starch degradation enzymes and their products were determined from chloroplasts and leaves of cotton (Gossypium hirsutum L. cv Coker 100) exposed t

5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 microM ionophore A23187 for 15 min at 37 degrees C in the presence of 50 mM L-serine released 69 +/- 28 and 1.5 +/- 0.8 (mean +/- SEM) ng of LTC4 and LTB4, respectively, per 10(6) cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 microM) or phorbol myristate acetate (1 microgram per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by L-serine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 +/- 3 ng per 10(6) cells) relative to LTB4 (6.0 +/- 3.1 ng per 10(6) cells), whereas neutrophils from the same donors released LTB4 (48 +/- 21 ng per 10(6) cells) in a greater than 7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.

A low-activation energy (35 000 cal.) for thermal inactivation and a large Km value (11·10−3 moles) for substrate affinity were found from carbonic anhydrase of the cotton plant, which was reported to be heat-tolerant but low in the rate of CO2-fixation.

Q-Enzyme is responsible for branching during the biosynthesis of amylopectin, the major form of starch in leaves. Its activity, however, cannot be determined accurately in the presence of the degradative enzymes, α-amylase and R-enzyme (the debranching enzyme). Q-Enzyme activity was measured by incubating with amylose primer and measuring changes in absorbance of the glucan-I2 complex. Maximum interference by α-amylase occurred early because it attacked the amylose primer. Maximum interference by R-enzyme occurred later during the incubation period, because R-enzyme affected branched glucan which was formed at the later stage of incubation. Therefore, these enzymatic interferences occurred in sequence at two separate times and sites. Dialysis of Q-enzyme preparation against EDTA greatly decreased the activity of α-amylase, probably by removing calcium. Addition of calcium to the dialyzed enzyme preparation restored most of the amylase activity. R-Enzyme was removed from the reaction mixture by complexing it with alumina (r)-gel. The insoluble complex was then removed by centrifugation. Dialysis of the enzyme extract against EDTA, followed by treatment with alumina (r)-gel, effectively overcame interferences by α-amylase and R-enzyme, and permitted accurate measurement of Q-enzyme activity.

Samples of polystyrene and poly(methyl methacrylate) have been prepared by bulk polymerization under conditions previously investigated in the literature. The molecular weight distributions were determined for early-conversion samples by gel permeation chromatography, and the experimental distribution curves were compared with calculated curves based on kinetic data. The agreement between the experimental and calculated curves was such as to indicate the utility of gel permeation chromatography in the investigation of polymer reaction kinetics and mechanisms.

A method for fluoride determination has been developed in which a level of 0.0–2.5 μg fluoride was determined quantitatively from several milligram samples of biological material. This technique allows the drying, ashing, and diffusion of small samples without transfer. The SPADNS-zirconium lake is a sensitive and convenient color reagent. Recoveries of 96 to 102% were obtained.

Methods are described for the isolation and characterization of the nucleic acids of Oenothera organensis pollen. Alkaline hydrolysis, followed by paper chromatography served to characterize the ribonucleic acid. However, the low concentration of deoxyribonucleic acid and the large amounts of interfering polysaccharide material required application of special techniques and column chromatographic methods. In addition, a method is described for the isolation of “native” deoxyribonucleic acid from root tips of Zea mays and Oenothera .

In many previous studies developing barley embryos have been shown to be highly radiosensitive. With acute irradiations, many of the responses exhibited are dependent in great measure upon the specific embryonic stage irradiated. The present study was undertaken to determine to what extent nucleic acid metabolism in barley embryos might be altered following acute irradiation, and whether here, too, embryonic stage would play a decisive role. Embryos developing within the ovaries of the parent plants were irradiated with 450 r acute X-radiation at one of four specific stages: early proembryo, late proembryo, mid-differentiation, or late differentiation. Fractional analyses of the individual bases of DNA and RNA were carried out at embryonic “maturity”. Because of the presence of interfering substances within the embryos, modification of previously published methods was necessary, the procedures developed being described herein. The DNA of control embryos is of the A-T type with the ratio between guanine and cytosine being unity and that between adenine and thymine being only 0·68. The total nucleic acid content of irradiated embryos showed residual depressions of 30–36 per cent, most of this being due to an effect upon RNA which makes up about 85 per cent of the total nucleic acid at embryonic maturity. Overall depression of RNA amounted to as much as 39 per cent but with a maximum difference among irradiated groups of only 5 per cent. This low variability in RNA content indicates that neither difference in embryonic stage nor post-irradiation time are important factors. In contrast, DNA depression ranged from 7 to 33 per cent among irradiated groups, the degree of depression being inversely correlated with the embryonic stage irradiated. With the exception of the oldest embryo group, residual DNA depression was independent of post-irradiation time. Of the RNA bases, guanine and cytosine showed proportionately less alteration than adenine (which was the least radiosensitive) or uracil (which was the most labile). Changes in DNA base components were generally more complex with wide fluctuations in the amount of cytosine and guanine depression. Effects on adenine were less pronounced. Of the DNA bases, thymine was characteristically the most labile, being the only DNA base to be significantly depressed in all four irradiated groups and the only one to be so affected in two of the groups. Embryos irradiated early in proembryo development or late in differentiation showed greater degrees of residual DNA depression at embryonic maturity but with little alteration in DNA base ratios. Irradiation during late proembryo development, in contrast, produced little lasting effect on DNA content, but marked changes in DNA base ratios primarily because of a decrease in thymine. When compared with results from other studies the present data suggest that a relationship exists between alteration of DNA base ratios and the occurrence of organ abnormalities during embryonic development. Metabolic disturbance as an expression of intra- or inter-tissue incompatibility mediated through radiation-induced genetic heterogeneity is considered as a possible cause.

Investigation was made for the effect of fluoride on plant growth, acid soluble nucleotides, and RNA in germinating corn seedling roots. Fluoride suppresses root growth as measured by changes in fresh weight. Column chromatographic analyses demonstrated that fluoride modifies ratios of acid soluble nucleotide species. The relative amount of nucleotides is altered mainly due to triphosphate nucleotides of which ATP is most accumulated. Paper chromatographic analyses showed that fluoride induces changes of RNA structure. The RNA is characterized by lowered relative content of cytosine and by increased ratio of cytosine to guanine. Adenine is depressed significantly only in the root tissue treated by the highest fluoride concentration.

A study was made of the relationship between the level of r RNA and the rate of growth in pinto bean plants treated with 0·35 ppm ozone for 20–40 min. Cytoplasmic ribosomes and a fraction containing both cytoplasmic and chloroplast ribosomes were initially isolated from primary leaves. The r RNA components of 25S, 23S, and 16S were extracted from these fractions and analysed by sucrose density gradient. The levels of cytoplasmic r RNA components, 25S and 16S, are not significantly affected by ozone. In contrast, ozone specifically decreases the relative level of 23S of chloroplast r RNA, as the rate of growth is inhibited.

A study was made of cytoplasmic and chloroplast ribosomes in the primary leaves of pinto bean plants exposed to ozone. The isolated ribosomes were analysed by sucrose density gradient. Ozone at the levels of 0·35 ppm for 20–35 min does not change the concentrations of various sedimenting particles of the cytoplasmic ribosomes. Ozone at similar levels, however, specifically decreases the population of chloroplast ribosomes per unit fresh weight of leaves. The distribution pattern of these chloroplast ribosomes is characterized by the low concentration of the fast-sedimenting polysome particles concomitant with the low magnitude of other slow-sedimenting components. The kinetics of ribosome populations during leaf growth demonstrates that ozone does not influence the daily levels of different ribosomal components of cytoplasmic ribosomes. However, ozone prematurely decreases the concentrations of polysomes and other components of chloroplast ribosomes below control level at the early stage of leaf development. These findings are discussed to explain initiation of the premature senescence caused by ozone.

A study was made of ribosomes and ribonuclease in the roots of corn seedlings aged by fluoride. Fluoride induced a reduction of ribosome content in which a decrease in polysome level was accompanied by an increase in number and concentration of monosomes and subunits combined. Fluoride increased the specific activity of ribonuclease, probably associated with ribosomal protein. This enhanced activity was likely due to enzyme activation caused by fluoride. Sucrose density gradient analyses demonstrated that ribosome fractions contained a mixture of heterogeneous RNA components.

Growth inhibition caused by fluoride was further studied by analyzing free and bound ribosomes, and the ribosomal components in corn roots. Ribosomes were isolated by differential centrifugation. Ribosomal components were analyzed by column chromatography and electrophoresis. Fluoride reduces the amounts of both free and bound ribosomes. Fluoride also modifies the RNA to protein ratio of bound ribosomes more than that of free ribosomes. Fluoride does not affect base ratios of the ribosomal RNA, but reduces its content and changes the structure of the ribosomal protein moiety.

Summary Chloroplast ribosomes and cytoplasmic ribosomes were isolated from the primary leaves of control and ozone-treated pinto bean plants. On the basis of a unit of ribosomal protein, the levels of ribosomal sulfhydryl groups and their kinetic responses to 5,5′-dithiobis(2-nitrobenzoic acid) were assayed by Ellman's procedure in the presence of sodium dodecyl sulfate. The sedimentation profile of ribosomes was determined by sucrose density gradient. Ozone, 0.30 ppm for 20 to 50 min, decrease the levels of sulfhydryl groups in chloroplast ribosomes far more than in cytoplasmic ribosomes. Both ozone and p-chloromercuribenzoate directly dissociate polysomes and monosomes of isolated chloroplast ribosomes into smaller particles in vitro. The levels of sulfhydryl groups which react with 5,5′-dithiobis(2-nitrobenzoic acid) are higher in chloroplast ribosomes than in cytoplasmic ribosomes. The reaction with 5,5′-dithiobis(2-nitrobenzoic acid) of this higher level of sulfhydryl groups in chloroplast ribosomes goes to completion much faster than the reaction of the lower level of sulfhydryl groups in cytoplasmic ribosomes. With these data, the mechanism which is directly involved in the previous finding, ozone's preferential influence on the disintegration of chloroplast ribosomes in bean leaves is discussed.

Nonaqueous fractionation of leaves of the cotton plant suggested that carbonic anhydrase was associated with the chloroplasts. Activity of this enzyme in aqueous extracts prepared in media containing no reductants was stable at 4°. Response to sulfhydryl reagents varied. The results indicated that thiol groups, necessary for the activity of the enzyme, were partially protected from oxidation.

Spinach plants were treated with 0.35 ppm ozone and the photochemical activity of photosystem II determined after separation from the isolated chloroplasts using digitonin. Ozone impaired the activity of the system and also reduced the amount of β-carotene in the chloroplasts.

The rates of incorporation of P/sup 32/ into RNA and DNA were stadied by autoradiography in different embryonic stages of barley. The total amount of incorporated P/sup 32/ increased during the embryonic development. The P/sup 32/ incorporation rate in DNA decreased gradually as the growth rate slowed down. The results indicate that RNA turnover in slow or nongrowing tissue is still relatively large. (D.L.C.)

Fluoride-polluted navel orange leaves, Citrus sinensis (Linn.) Osbeck, were fractionated into the subcellular components in hexane/carbon tetrachloride mixtures having various densities. Fluoride was determined at each fraction. Analyses were also made for the subcellular distribution of chlorophyll, nitrogen, and DNA to assess the extent of cross-contamination of each component. The fraction containing cell wall, nuclei, and partly broken cells apparently contained a major amount of fluoride. However, if allowance was made for the cross-contamination of chloroplasts and chloroplast fragments, the fraction of chloroplasts was found to be the site of the highest fluoride accumulation. When each particulate component was washed with water after drying, the combined washings contained more than 50% of the total fluoride of the isolated fractions. The usual method of subcellular fractionation with aqueous solvent shifted the major site of fluoride accumulation from the fraction of chloroplasts to that of the supernatant.

Cotton plants (cv. Coker 100) were exposed to a 14-hour dark period. Starch degradation occurred with no accumulation of sugars due mainly to translocation. Considerable amounts of starch degradation products however were detected from leaves after phloem transport was blocked. A minor component (10 to 25% of total starch) with a linear structure, amylose, was preferentially degraded, whereas the major multiple-branched component (about 80%), amylopectin, showed an increasing resistance to degradation with leaf age. This relationship was also shown by the decreasing iodine-binding capacity of unit starch with increasing leaf age. The structural resistance of amylopectin to enzymic dark degradation was one of the barriers to starch dissolution in cotton.

During cotton leaf development, starch accumulation was characterized by an initial rise to a maximum at the second to the fourth leaf from the apex. Then, starch content progressively decreased with leaf age. Starch accumulation was inversely related to the ratio of amylopectin to amylose. Differences between leaves in this ratio resulted from variations in both amylose and amylopectin levels. Fluctuations in amylose levels were more extreme than those of amylopectin. During the diurnal cycle, amylopectin was accumulated more than amylose in both young and old leaves during the day. During the night, amylopectin was degraded more than amylose in young leaves and vice versa in older leaves. The rate of change of the amylopectin to amylose ratio during the day was consistently higher than that during the night.

A method is described for manometric determination of bicarbonate (as little as 0.25 μmoles) in leaves. Conditions such as optimum pH and inactivation of carbonic anhydrase are introduced to stabilize the bicarbonate. Crude leaf extracts are purified with chloroform and Darco G-60. An ionic mixture is separated from macromolecular compounds by elution from a column of Sephadex G-25. Carbon dioxide is released by acidifying the ionic mixture and is determined with a respirometer.

A procedure was developed for the separation and purification of amylose and amylopectin isolated from cotton leaves. Cotton leaves were homogenized in 0.02 molar phosphate buffer at pH 7.0 containing HgCl(2) plus toluene. Crude starch granules were collected by centrifugation and partially purified by treating with acetone and toluene. The starch granules were then dispersed in dimethylsulfoxide and precipitated with ethyl alcohol. The precipitate was suspended in boiling water. Amylose was separated from amylopectin and cell wall particles on a Sepharose 2B column and further purified with thymol and butanol. Amylopectin was then separated from the colloidal cell wall contaminants by its specific interaction with concanavalin A. Purities of starch components were verified by specific biochemical and enzymic tests in addition to their iodine-binding capacity. This procedure should also be suitable for purification of starch components from other plant sources.

Leukotriene C 4 (LTC 4 ) was metabolized by human polymorphonuclear leukocytes (PMNs) stimulated with phorbol myristate acetate (PMA) into three sets of products. These products differed in mobility on reverse-phase high-performance liquid chromatography (RP HPLC) from LTC 4 and also from leukotriene D 4 (LTD 4 ) and leukotriene E 4 (LTE 4 ), the sequential products of peptide cleavage of LTC 4 . Products I, II, and III were eluted as doublets with an average retention time for each doublet of 7.5 ± 0.3, 10.5 ± 0.6, and 16.3 ± 1.1 min (mean ± SD), respectively, as compared with 13.8 min for LTC 4 . Doublet I material was biologically inactive and showed 4 , doublet II material had 1% of the spasmogenic activity of LTC 4 on the guinea pig ileum and was equally immunoreactive, and doublet III material was neither biologically active nor immunoreactive. When [14,15- 3 H]LTC 4 and [ 35 S]LTC 4 were metabolized, all three doublet products retained the 3 H label, whereas only the doublet I and doublet II products retained the 35 S label. The UV absorbance spectra of the three sets of metabolites were as follows: doublet I, maximum at 280 nm with shoulders at about 270 and 290 nm; doublet II, maximum at 284.5 nm with shoulders at about 275 and 295 nm; and doublet III, maximum at 269 nm with shoulders at about 259 and 279 nm. The metabolism of LTC 4 to the three classes of functionally inactive products by stimulated PMNs was completely blocked by catalase and azide, indicating a requirement for H 2 O 2 and myeloperoxidase. When hypochlorous acid (HOCl)—considered to be a natural product of the interaction of myeloperoxidase, H 2 O 2 , and chloride ion—was formed chemically and allowed to react with LTC 4 , the resulting products were indistinguishable by UV and HPLC analyses from the doublet II and doublet III metabolites of LTC 4 . The doublet II products were identified as the two diastereoisomeric sulfoxides of LTC 4 by comparison with synthetic reference compounds. The doublet III products were shown to be identical with synthetic samples of (5 S , 12 S )- and (5 S , 12 R )-6- trans -LTB 4 . The formation of two diastereoisomeric LTC 4 sulfoxides and 6- trans -LTB 4 can be explained in terms of an S -chlorosulfonium ion as the initial reactive intermediate, which subsequently undergoes conversion to product II by hydrolysis and product III by carbocation formation.

Little attention has been given to the occurrence of exocellular hydrolytic enzymes associated with the roots of higher plants. Such enzymes, if present, could solubilize macromolecular constituents of organic soils, permitting uptake and utilization by the plant of the resultant low molecular weight organic compounds. The present work supports this concept by demonstrating that several exocellular hydrolytic enzymes are associated with the roots of intact corn seedlings. Distinction has been made between exocellular enzymes released into the culture media and enzymes apparently bound to the root surface. A previous paper from this laboratory (17) reviewed earlier studies and presented evidence for the occurrence of exocellular enzymes associated with the primary cell wall of corn coleoptiles. Of particular relevance to the present work is a report (36) of the secretion of peroxidase, acid phosphatase, indolylacetic acid oxidase, and amylase into the culture media bathing plant *tissue cultures. In addition, reports have appeared (2, 6, 14) of the occurrence of an exocellular invertase, possibly involved in sucrose metabolism by roots. In the present work it is reported that invertase, cellobiase, adenosine triphosphatase, pyrophosphatase, and nuclease activity is observed when appropriate substrates are added to nutrient solution bathing the roots of intact seedling corn plants. In addition, inveritase and nuclease are detected as soluble enzymes in the nutrient solution.