Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.     

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.     

Increased superoxide radical production evokes inducible DNA repair in Escherichia coli

Paraquat induced the SOS response in Escherichia coli. This was measured in terms of acquired resistance towards UV lethality in a wild-type strain and in terms of appearance of beta-galactosidase activity in a din::Mu d(Ap lac) fusion strain. However measured, the induction of the SOS response by paraquat was entirely dioxygen-dependent; whereas induction of the SOS response by mitomycin C was independent of the presence of dioxygen. As expected, recA(Def) and lexA(Ind-) isogenic strains did not show the SOS response. It appears likely that O-2, whose intracellular production is increased by paraquat, leads to DNA damage which in turn induces the SOS response.     

The effects of colchicine analogues on the reaction of tubulin with iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide)

We have previously found (Ludue?a, R. F., and Roach, M. C. (1981b) Biochemistry 20, 4444-4450) that colchicine and podophyllotoxin inhibit the alkylation of tubulin by iodo[14C]acetamide and the formation of an intrachain cross-link in the beta-tubulin subunit by N,N'-ethylenebis(iodoacetamide) (EBI). It was not clear whether these effects were due to conformational changes in tubulin induced by drugs or to direct steric blockage of the sulfhydryl groups involved. In an effort to characterize further these phenomena, we have examined the effects of single-ring and bicyclic analogues of colchicine on the reaction of tubulin with iodo[14C]acetamide and EBI. We have found that neither the A-ring analogues, 3,4,5-trimethoxybenzyl alcohol, 3,4,5-trimethoxybenzaldehyde, 2,3,4-trimethoxybenzaldehyde, and benzaldehyde, nor the C-ring analogues, tropolone and tropolone methyl ether, inhibited alkylation. In contrast, colchicine, podophyllotoxin, and nocodazole and the bicyclic analogues, 5-(2',3',4'-trimethoxyphenyl)-2-methoxytropone and combretastatin, inhibited tubulin alkylation. Since the presence of a bond joining the A and C rings seems to be the determining factor in the suppression of alkylation, it is likely that inhibition by colchicine of the reaction with iodo[14C] acetamide is due largely to a conformational change induced by colchicine. A different pattern was obtained when the effects on cross-link formation by EBI were examined. Here, all the A-ring analogues, the bicyclic analogues, and colchicine, podophyllotoxin, and nocodazole all inhibited formation of the cross-link, whereas the C-ring analogue tropolone methyl ether did not inhibit cross-link formation. Since compounds whose effect on alkylation is markedly different have the same effect on cross-link formation, it is possible that this effect is a steric one and that perhaps the A-ring of colchicine binds to tubulin very close to one of the sulfhydryls involved in the intrachain cross-link formed by EBI in beta-tubulin.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

Enzymes of vitamin B6 degradation. Purification and properties of two N-acetylamidohydrolases

alpha-(N-Acetylaminomethylene)succinic acid hydrolase (Compound A hydrolase, EC 3.5.1-) and alpha-hydroxymethyl-alpha'-(N-acetylaminomethylene)succinic acid hydrolase (Compound B hydrolase, EC 3.5.1-) were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. The two inducible enzymes catalyze Reactions 1 and 2, respectively, which release the first generally useful anabolic intermediates during growth of these organisms with (formula; see text) pyridoxine as a sole source of carbon and nitrogen. Compound A hydrolase is a monomeric protein of Mr 32,500 with aspartic acid as its NH2-terminal residue. Compound B hydrolase (Mr congruent to 205,000) is a multimer containing probably six identical subunits with glycine as the NH2 terminus. The two enzymes have quite different amino acid analyses, although both are high in Asx and Glx, lack tryptophan, and show similar stabilities to pH and temperature. Compound A hydrolase has a pI of 4.4, a Km of 3.3 microM, and a Vmax of 3.1 mumol X min-1 X mg-1 at pH 6.5 and 25 degrees C; no analogue substrates were found. Compound B hydrolase has a pI of 4.2, a Km of 25 microM, and a Vmax of 3.8 mumol X min-1 X mg-1 at 25 degrees C and pH 7.0; it also hydrolyzes Compound A slowly. Both enzymes are inhibited competitively by di- and tricarboxylic acids, itaconic acid being among the most effective. Sulfite inhibits both enzymes by a time-dependent mechanism not yet understood. The two amidases appear to differ greatly in architecture despite the similarity in properties and in the overall reactions they catalyze.     

Cytogenetic and blood group studies of sheep/goat chimaeras

Aggregation chimaeras were composed of quarter (or 1 cell) contributions from 4-cell blastocysts of sheep or goats, or of an 8-cell blastocyst of one species enveloped in three 8-cell blastocysts of the other. Gestation was in sheep or goat recipient females. Of the 10 living animals born, 3 were identified as interspecific chimaeras by body conformation and coat type among the 7 quarter/quarter aggregations and 1 among the 3 giant aggregates. Interspecific chimaerism was identified by cytogenetic study of umbilicus and blood lymphocytes respectively of 2 of these, one from each type of aggregate. Intraspecific sex chimaerism was found in 3 other animals; 2 were of giant aggregate origin, but the 1 of quarter/quarter origin must have acquired it by placental anastomosis with a twin conceptus. Tests using species-specific monoclonal antibodies and electrophoretic separation of haemoglobins and isoenzymes demonstrated sheep and goat erythrocytes in one giant aggregate chimaera; their relative proportions and those of the blood lymphocytes changed over a period of 31 months from approximately 60% goat and 40% sheep to more than 90% sheep. The plasma transferrins and amylases did not show similar relative changes from their predominantly goat-like character and, by implication, neither did their tissues of origin.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

FSH stimulation of cytosolic protein synthesis in cultured pig Sertoli cells

The effects of FSH on the cytosolic protein synthesis by a primary culture of immature porcine Sertoli cells were studied. Cells were cultured in a chemically-defined medium for 3 days and, on day 3, they were incubated for 40 h in another medium containing labelled amino-acids either in the presence or absence of 50 ng/ml porcine FSH (pFSH). In control cells, about 107 spots (pI in the range of 5 to 8) were identified by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled cytosolic proteins. pFSH treatment produced a marked increase of seven proteins whose molecular weights and isoelectric points are respectively comprised between 25 to 58 K and 5.5 to 5.8. In addition, pFSH treatment induced a slight but constant increase of two other proteins (mol. wt: 24 and 12 K and isoelectric point: 5.3).