SOD Type Research Articles

A novel superoxide dismutase gene encoding membrane-bound and extracellular isoforms by alternative splicing in Caenorhabditis elegans.

We have identified a novel Cu/Zn superoxide dismutase gene (termed SOD-4) in Caenorhabditis elegans. Characterization of its complementary DNA revealed that the gene encodes two isoforms by alternative splicing, SOD4-1 and SOD4-2 which differ in their C-terminal exons. Their predicted amino acid sequences include a consensus signal peptide at their N-termini and are homologous to the extracellular-types of Cu/Zn superoxide dismutase in mammals. In addition, SOD4-2 possesses a putative transmembrane domain at the C-terminal region. When transiently expressed in Chinese hamster ovary cells, both types were found in the membranes and SOD4-1 also in the culture fluid. It is, therefore, indicated that SOD4-1 is an extracellular form and SOD4-2 a membrane-bound form, the latter representing a novel type of SOD. In C. elegans, SOD4-2 mRNA was found to be preferentially expressed in eggs.

Preventive Effects of Superoxide Dismutase Derivatives Modified with Monosaccharides on Reperfusion Injury in Rat Liver Transplantation

Background.Superoxide dismutase (SOD) derivatives modified with monosaccharides such as mannosylated SOD (Man-SOD) and galactosylated SOD (Gal-SOD) are taken up by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis. We examined the preventive effects of Man- or Gal-SOD on cold ischemia–reperfusion injury in orthotopic liver transplantation (OLT) in rats.Methods.Male Lewis rats underwent OLT. Native SOD and modified SOD with mannose (Man-SOD) or galactose (Gal-SOD) were intravenously given just prior to reperfusion of the grafted liver.Results.After OLT, no significant changes in serum levels of aspartate aminotransferase or lactate dehydrogenase were evident among rats, with or without various types of SOD. However, tissue levels of Na+,K+-ATPase, an indicator of tissue viability, were significantly higher in rats treated with Man- and Gal-SODs compared with that in untreated OLT rats. When comparisons were made between Gal-SOD and Man-SOD, higher levels of Na+,K+-ATPase were evident in rats treated with Gal-SOD. Elevations of tissue levels of lipid peroxides were significantly inhibited by modified SODs. The survival rates of rats following OLT after prolonged cold preservation correlated with results of graft viability.Conclusions.SOD derivatives, particularly Gal-SOD, have protective effects on cold ischemia–reperfusion injury during OLT in rats.

Manipulating freezing tolerance in transgenic plants

Winterhardiness is a composite of tolerances to freezing, desiccation, ice-encasement, flooding and diseases. From one point of view, winterhardiness may not be easily manipulated by genetic engineering technology because many different genes are involved in the tolerance of these diverse stresses. However, these various stresses have similarities. They promote formation of activated forms of oxygen, promote membrane lipid and protein degradation, cause similar biophysical changes in membrane structure, and culminate with increased leakage of cytoplasmic solutes and loss of cellular membrane functions. These similarities led to the hypothesis that winter injury might be reduced in crop plants if their tolerance of oxidative stress was increased. Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress either Mn-SOD or Fe-SOD cDNA (provided by Dirk Inze, Universiteit Gent). Petiole explants were transformed using Agrobacterium tumefaciens and plants were regenerated by somatic embryogenesis. The primary transgenic plants were screened using PCR (polymerase chain reaction), Southern hybridization and native PAGE for SOD activity. Greenhouse and laboratory studies showed a minimal difference in stress tolerance between the primary transgenic and non-transgenic plants. In the first field trial, four primary transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival after two winters than the non-transgenic RA3. Similar results were obtained in a second field trial, comparing 18 independent transformants with Mn-SOD targeted to the mitochondria, 11 independent transformants with Mn-SOD targeted to the chloroplast and 39 independent transformants with Fe-SOD targeted to the chloroplast, expressed in three different non-transgenic plants. The transgenic plants averaged over 25% higher survival than the non-transgenic controls after one winter. There was no effect of subcellular targeting or SOD type on field survival, but there was variation among independent transformants containing the same SOD construct. Activated oxygen therefore appears to be one of the possible causes of winter injury, and it should be possible to reduce winter injury in transgenic plants by constitutive overexpression of SOD.

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases.

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.

Efficacy of Recombinant Human Manganese Superoxide Dismutase Compared to Allopurinol in Protection of Ischemic Skeletal Muscle Against “No-Reflow”

A growing body of experimental data indicates that the "no-reflow" phenomenon is a type of reperfusion injury in skeletal muscle which may, in part, be mediated by oxygen free radicals, and thus may be attenuated by using agents that scavenge or inhibit formation of these reactive oxygen metabolites. This study was undertaken to assess the efficacy of recombinant human manganese superoxide dismutase (rhMnSOD) in reducing reperfusion injury in skeletal muscle. The specific advantage of this agent over other SOD types is a much longer plasma half-life (5 to 7 hr), allowing better equilibration between extra- and intracellular compartments. The rat cremaster model was used to study "no-reflow" in skeletal muscle. Reperfusion injury in the muscle was assessed by fluorescein dye perfusion, myocyte creatine phosphokinase (CPK) release, and contractile function in response to electrical field stimulation. Compared with untreated saline control animals, those treated with rhMnSOD after 5 hr of cremasteric ischemia, had a significantly higher percentage area of blood reflow (78 percent +/- 6 percent of normal), a greater percentage tetanic (66 percent +/- 9 percent of normal) and twitch (56 percent +/- 9 percent of normal) contractile strength, and less CPK release (21.5 percent higher than pre-reperfusion baseline CPK levels) (p < 0.05). Untreated saline control CPK release (21.5 percent higher than the prereperfusion level. Animals treated with allopurinol also had a significantly higher percentage twitch contraction (47 percent +/- 14 percent of normal) and a lower CPK release (11.1 percent of the prereperfusion value) 45 min after reperfusion than untreated saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the addition of methylene blue to the reaction mixture for the hydrothermal synthesis of aluminophosphate molecular sieves on their morphology

The morphology of aluminophosphate hosts prepared by hydrothermal synthesis with the addition of methylene blue (MB) to the reaction mixture was compared with that of dye-free samples by means of adsorption and electron microscopy methods. With AIPO 4-5, SAPO-5, and FAPO-5 (AFI structure type), no effect of the addition of MB has been found. With AIPO 4-l1 (AEL structure type), MB has caused a 50% contamination with some nonadsorbing material. With AIPO 4-20 and SAPO-34 (SOD and CHA structure types), MB strongly influences the morphology of crystals.

Iron- and manganese-containing superoxide dismutases from Methylomonas J: identity of the protein moiety and amino acid sequence

Mn-superoxide dismutase (SOD) and Fe-SOD were isolated from Methylomonas J, an aerobic methylotrophic bacterium, grown in methylamine media containing either manganese (Mn-rich medium) or iron (Fe-rich medium), respectively. The specific activity of the Mn-SOD was 2250 units mg-1 (mol of Mn)-1 (mol of dimer)-1, and the metal content of the enzyme was 0.98 mol of Mn and 0.12 mol of Fe per mole of dimer, while those of Fe-SOD were 88.5 units mg-1 (mol of Fe)-1 (mol of dimer)-1 and 1.04 mol of Fe and 0.02 mol of Mn. The electrophoretic mobilities in the presence of sodium dodecyl sulfate, with or without urea, and the chromatographic behavior on an HPLC column using an octadodecyl silicated column and a gel permeation column were identical. Amino acid compositions were practically indistinguishable in both SODs. The enzyme activity was restored by dialysis of an apoprotein obtained from the Mn-enzyme with either manganese sulfate or ferrous ammonium sulfate up to an activity level similar to that for the native Mn-SOD and the native Fe-SOD, respectively. The same result has been reported with the reconstitution using an apoprotein obtained from the Fe-enzyme [Yamakura, F., Matsumoto, T., & Terauchi, K. (1990) Free Radical Res. Commun. (in press)]. These results suggest the possibility that both types of SODs are composed of a single apoprotein synthesized in cells grown in either the Fe-rich medium or the Mn-rich medium.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR method for superoxide dismutase assay in brain and liver homogenates

A method for copper- and manganese-containing superoxide dismutase (Cu- and MnSOD) assay in tissue homogenates such as liver and brain, based on the measurement of the longitudinal nuclear relaxation time ( T 1) of F −, has been develped as a preliminary approach to in vivo measurement of these enzymes. The relaxation rate of F −1, which increases linearly with the SOD concentration, also depends on the oxidation state of the metal ion present in the active site of the enzyme. The relaxivity values of the oxidized, reduced and turnovering CuSOD were found to be 9.6×10 6, ⪡ 1×10 2 and 5.2×10 6 M −1 s −1, respectively, while for MnSOD the corresponding values were 2.9×10 6, 4.2×10 6 and 3.6×10 6 M −1 s −1, respectively. These high relaxivity values allow the detection of SODs in brain and liver homogenates where, under aerobic conditions, these enzymes appear in the steady-state. The contribution of the two types of SOD to the F − relaxation rate in the homogenates was measured by addition of either diethyldithiocarbamate or cyanide, both of which selectively inhibit the CuSOD. The comparison between NMR and activity data confirmed the possibility of carrying out accurate and precise measurements of SODs in homogenates by NMR.

Targeting Sod by Gene and Protein Engineering and Inhibition of Free Radical Injury

Although oxygen toxicity of tissues can be decreased by a variety of antioxidants and some enzymes, such as SOD and catalase, their protective effect on tissue injury in various diseases are fairly small predominantly because of their unfavorable in vivo behavior. To minimize oxidative stress in various diseases, such as ischemic myocardial injury, circulatory disturbance and corneal inflammation, we synthesized three types of SOD derivatives by gene and protein engineering technique. One type of SOD (SM-SOD covalently linked with hydrophobic anions) circulates bound to albumin with a half life of 6 h and accumulates in tissues whose local pH is decreased. The other type of SOD (AC-SOD covalently linked with long chain fatty acids via the epsilon-amino group of lysyl residues) anchors onto membrane/lipid bilayers of various cells. The last type of SOD (HB-SOD synthesized by constructing a fusion gene coding human CuZn-type SOD and a C-terminal heparin-binding domain) binds to heparin-like proteoglycans on vascular endothelial cell surface. Intravenous administration of either SM-SOD or HB-SOD markedly inhibited postischemic reflow arrhythmias in the rat. When the left anterior descending artery was occluded permanently, about 65% of animals died within 30 min predominantly due to irreversible ventricular fibrillation; the motality of animals decreased to 15% by administering SM-SOD either before or after occlusion. Topically administered AC-SOD bound to the corneal epithelial cell surface and polymorphonuclear leukocytes and efficiently dismutated superoxide radicals at their cell surface. Thus, endotoxin-induced keratitis was inhibited markedly by topical instillation of AC-SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Contamination of adenosine deaminase by superoxide dismutase: Stabilization of endothelium-derived relaxing factor

We have measured cyclic GMP accumulation in co-cultures of bovine aortic endothelial cells and rat smooth muscle cells as an index of endothelium-derived relaxing factor (EDRF) production. Adenosine deaminase (EC 3.5.4.4, Sigma type VI) produced a 5- to 10-fold increase in the basal and bradykinin-stimulated cyclic GMP content of co-cultures but had no effect on smooth muscle cells alone. Cyclic GMP accumulation in response to adenosine deaminase was not blocked by adenosine deaminase inhibitors or affected by adenosine, the products of adenosine deamination (inosine and ammonia), or adenosine receptor antagonists. Since Superoxide anion is known to destroy EDRF and nitric oxide (NO) (which is similar or identical to EDRF in composition), we tested for Superoxide dismutase (SOD, EC 1.15.1.1) in single lots of eight commercial sources of adenosine deaminase by measuring inhibition of the superoxide-mediated reduction of cytochrome c. SOD activity was found in all sources of adenosine deaminase, but varied widely. One lot of Sigma type VI enzyme contained 0.08 units SOD/ unit adenosine deaminase. The ec 50 values of purified SOD (0.23 units/mL) and Sigma type VI adenosine deaminase (2.1 units/mL) needed to increase the cyclic GMP content of co-cultures differed by a similar factor, 0.11. Thus, the SOD activity in adenosine deaminase is sufficient to account for its effect on cyclic GMP accumulation. One lot of Boehringer Mannheim adenosine deaminase contained much less SOD contamination (0.006 units SOD/unit adenosine deaminase) and produced much less accumulation of cyclic GMP in co-cultures. Cyclic GMP accumulations in response to adenosine deaminase and SOD were both abolished by the NO synthetase inhibitor N G - monomethyl- l - arginine (0.1 mM), consistent with the idea that these enzymes act by stabilizing EDRF. Adenosine deaminase and the SOD activity contaminating it were found to have similar molecular masses of 33–34 kD as assessed by gel permeation chromatography. When run under reducing conditions to dissociate homodimeric SOD into monomers, a 16.6 kD peptide which co-migrates with purified cupro-zinc SOD was visible in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the Sigma type VI but not the Boehringer Mannheim adenosine deaminase. We conclude that commercial sources of adenosine deaminase are variably contaminated by SOD. Since EDRF is synthesized by many tissues, the use of adenosine deaminase contaminated with SOD may produce numerous effects not attributable to the deamination of adenosine.

Comparative study of oxygen toxicity in human fibroblasts and endothelial cells

The resistance of human pulmonary fibroblasts (WI-38) and human umbilical vein endothelial cells to oxygen toxicity (1 atm O2) was compared. Endothelial cells were more sensitive than fibroblasts. They contained also less antioxidant enzymes except for SOD: respectively 132%, 96%, 70%, 59%, and 21% of the SOD, GSH peroxidase, GSH reductase, catalase, and G6PD content of fibroblasts. However, they contained 1.81-fold more GSH than fibroblasts. Their lower content of antioxidant enzymes can explain their higher sensitivity to oxygen. The efficiency of natural antioxidant molecules and enzymes in the protection of cells incubated 3 days under 1 atm O2 was studied. alpha-tocopherol added in the culture medium led to a significant protection, contrary to the result for ascorbic acid. Microinjection of catalase, SOD, and GSH peroxidase directly into the cells was also tested: the protection was concentration dependent for both types of cells but SOD did not protect the endothelial cells. Lower activities of the other enzymes were needed to achieve protection of the endothelial cells, compared to fibroblasts. Since endothelial cells were also shown to display lower antioxidant enzyme activities, it can be hypothesized that their content is optimized for survival in physiological conditions.

Leucocyte superoxide dismutase levels in acute and chronic leukemias

Superoxide dismutase “SOD” enzyme levels in leucocytes of patients with acute and chronic leukemia, were measured by using Misra and Fridovich's epinephrine method. In all types of leukemia, the SOD levels were found to be significantly higher than normals. However, these levels decreased to normal in remission. No relationship was found between the SOD level and types of leukemia. The absence of relationship between the increase in SOD level and various clinical parameters suggests that this increase is the result of a disturbed gene expression in the stem cells. Hence it is of interest to ascertain the effect of the chemotherapy leading to the normalization of the SOD levels.

Superoxide dismutase in epidermis (1).

AbstractSuperoxide dismutase (SOD), which catalytically scavenges superoxide anion (O–2), constitutes an essential defense against the toxicity of oxygen. We investigated the enzyme activity of pig skin epidermis. SOD activity was determined by monitoring the inhibitory effect of SOD on red formazan formation from neotetrazolium, which depends on (O–2) generation. (O–2) was generated by the hypoxanthine‐xanthine oxidase reaction. Pig epidermis contained significant amounts of heat‐labile SOD activity which was proportional to the added epidermal homogenate. The optimal pH of the reaction was between pH 8.2 and 8.5. Metallochelating agents such as cyanide, sodium azide, and diethyldithiocarbamate (DDC) inhibited the epidermal SOD activity in a dose‐dependent manner. It has been known that two types of SOD, a Cu, Zn‐type and a Mn‐type, are present in eukaryotes; that the latter is insensitive to cyanide inhibition. Using this property, the main SOD present in the epidermis was hypothesized to be the Cu, Zn‐type (8.6 ±1.1 unit/mg protein; around 75%); the Mn‐type was a minor component (2.8 ± 0.2 unit/mg protein; around 25%). SOD staining following acrylamide disc gel electrophoresis revealed two epidermal SOD bands, one of which was abolished by the addition of cyanide. These results are consistent with the view that pig epidermis contains two types of SOD, a Cu, Zn‐type and a Mn‐type; the former appears to be predominant.

Solid state transformations of synthetic CHA-and EAB-type zeolites in the sodium form

The sodium forms of CHA- and EAB-type zeolites (having 6-ring sequences of AABBCC and ABBACC respectively), transform topotactically to SOD type products above 600°C in dry N 2. The temperatures of transformation depend greatly on aluminium content, the number of protons and the amount of water present. Principal features of these frameworks are that they contain double 6-rings and that one 6-ring layer separates the transforming layers. X-ray Rietveld analyses of Na exchanged CHA- and EAB-type zeolites at various temperatures indicate that initially T′ O T bridges connecting the 6-rings of the hexagonal prism are ruptured. The mechanism of transformation involves the breaking of two T′O T bridges by a catalytic reaction of water, and pivoting of T′ about the remaining T′O T bridges leading to inversion of T′O 4 tetrahedra.

THE EFFECT OF SOD TYPE ON THE OCCURRENCE OF GROUND BEETLES (COLEOPTERA: CARABIDAE) IN A PEST MANAGEMENT APPLE ORCHARD

AbstractThe effects on carabids of natural, fescue, and rye sod types and of tillage were investigated in a pest management apple orchard. Carabids were sampled before and after the treatments by pitfall trapping and by two types of soil sampling. There were no significant effects of sod type on pitfall trap catches; however the abundance of all common species in soil samples was significantly affected by sod types. Usually in soil samples carabids were most abundant in natural sod and least abundant in tilled plots; numbers were intermediate in fescue and rye. Sod type did not affect structure or diversity of the carabid fauna.

The effect of trace elements on stock liveweight gain on West Coast soils

Abstract The results of trials investigating lamb and calf live weight responses to the trace elements selenium, copper, and cobalt are reported. Twenty-seven trials involved Se eight trials involved Cu, and four trials involved Co. When the trials were grouped on the basls of sod type the only concluslve result was a significant calf live weight gain response to Se on recent soils. However, there was a large variation in treatment effect between trials within soil types, and more trial work is required before discarding the posslblhty that effects other than soil type may exist.

Effects of environmental and cultural practices on calcium nutrition of fruit trees

Abstract The effects of climate, soil, management, fertilization, rootstocks, and cultivars on calcium concentration in the leaves were investigated in long term experiments in southern, central or eastern Poland. The concentration of Ca in the leaves of apple trees on heavier soil with higher rainfall was higher than on sandy soil and dry climate. McIntosh/M 11 showed a higher level of Ca in the leaves than on Antonovka seedlings, while in the cv. Schatenmorelle sour cherry the effect of rootstock was negligible. There was a definite influence of cultivar in the same fertilizer treatments on Ca accumulation in the leaves. Cultivar Bancroft always showed the lowest level of Ca in comparison with Mclntosh and Jonathan. Higher doses of fertilizers usually lowered the level of Ca in the leaves of younger apple trees (Nowa Wies), but not in the old trees (Dabrowice). The accumulation of Ca in the leaves of apple trees was not affected by the type of orchard soil management, clean cultivation, or sod.

Trends of Nonstructural Carbohydrates in the Stem Bases of Switchgrass

penetration, distribution and activity in southern grasses measured by yields, drought symptoms and P32 uptake, Agron. J. 46:229-233. Burton, G. W., J. E. Jackson, and R. H. Hart. 1963. Effects of cutting frequency on yield, in vitro digestibility, and protein, fiber and carotene content of coastal Bermudagrass. Agron. J. 55:500-502. Burton, G. W., and J. E. Jackson. 1962. Effect of rate and frequency of applying six nitrogen sources on coastal Bermudagrass. Agron. J. 54:40-43. Burton, G. W., and J. E. Jackson. 1962. Single vs split potassium application for coastal Bermudagrass. Agron. J. 54:1 3-14. DeVane, E. H., M. Stelly, and G. W. Burton. 1952. Effect of fertilization and management of different types of Bermudagrass sods on the nitrogen and organic matter content of Tifton sandy loam. Agron. J. 44:176-179. Hojjati, S. M., R. A. MeCreery, and W. E. Adams. 1968. Effects of nitrogen and potassium fertilization, irrigation and clipping interval on chemical composition of coastal Bermudagrass (Cynodon dactylon (L.), Pers.). I. Total available carbohydrates. Agron. J. 60:617-619. Jones, J. B., Jr., and M. H. Warner. 1969. Analysis of plant ash solution by spark emission spectroscopy, p. 152-160. In E. L. Grove and A. J. Perkins (ed.), Development in applied spectroscopy. Vol. 7A, Plenum Press, New york, King, W. A., C. C. Brannon, and H. J. Webb. 1961. Comparison of coastal Bermuda and oat hays with alfalfa hay for milking cows. S. Carolina Agr. Exp. Sta., Clemson Univ., Bull. 490. 30 p. Longstaff, W. H. 1963. Soil fertility management of coastal Bermudagrass on the upland sandy soils of east Texas. Hoblitzelle Agr. Lab., Texas Res. Found. Bull. 17. 27 p.

Postharvest Cultural Practices Affecting the Rooting of Kentucky Bluegrass Sods Grown on Organic and Mineral Soils1

AbstractThe effects of sod type, fertilizer placement, underlying soil type, soil moisture, sod harvest depth, and the postharvest irrigation rate on the transplant rooting capability were investigated. Market‐quality Merion Kentucky bluegrass (Poa pratensis L.) sod, grown on organic and mineral soils, was transplanted to grass‐faced root observation boxes. Root production data and counts of roots visible on the glass face were collected. Harvesting sod at a standard 2‐cm depth of cut was superior to 1‐cm in terms of root growth after transplanting. The 1‐cmthick sod was more prone to desiccation injury. Transplanting sod onto sandy loam topsoil of a 1:1 topsoilsubsoil mix resulted in increased root production compared to a clay loam subsoil. Transplanting sod to a dry soil resulted in delayed rooting. No difference in root production between soil‐incorporated and sod surface fertilizer placement was found in these short‐term studies. Sod grown on organic soil produced more roots in 16 to 24 days following transplanting than sod grown on mineral soil. Transplant sod rooting was impared during the May‐June period of extensive seedhead development.

Effect of Nitrogen, Phosphorus and Potassium Fertilizer on the Economics of Producing Bluegrass Forage1

SynopsisResponses of a native bluegrass sod to topdressed applications of N, P and K fertilizer rates varying jointly were used to fit a forage yield equation and to calculate iso‐yield curves and least cost fertilizer ratios. It was then shown how these data and equations may be used in determining levels of fertilizer for optimum beef production and estimating the price of beef needed to justify fertilization of this type of sod.