An alkaline proteinase of an alkalophilicBacillus sp.

An alkaline serine proteinase was purfied from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight was estimated to be 22,000 by a gel filtration method and 31,000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 8.2. The amino acid composition and CD spectrum were determined. The alkaline proteinase had a pH optimum at 10–11 for milk casein digestion. The specific activity of the alkaline proteinase was 0.35 katal/kg of protein at pH 10.0 for milk casein hydrolysis.The substrate specificity of the alkaline proteinase was studied by using the oxidized, insulin B-chain and angiotensin. An initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Leu¹¹-Val¹², and additional sites at Gln⁴-His⁵, Tyr²⁶-Thr²⁷, and Asn³-Gln⁴ in the oxidized insulin B-chain at pH 10.0. In comparison with the subtilisins Carlsberg and Novo, the alkaline proteinase fromBacillus sp. showed a unique specificity toward the oxidized insulin B-chain. Hydrolysis of angiotensin at pH 10.0 with the alkaline proteinase was observed at Tyr⁴-Ile⁵. The proteinase has aK _m of 0.1 mM andk _cat of 3.3 s^–1 with angiotensin as substrate.     

Specificity of Thermophilic Streptomyces Alkaline Proteinase

The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe²⁴-Phe²⁵, Leu¹⁵-Tyr¹⁶ and Leu¹¹-Val¹²) and additionally at six others (Leu⁶-CySO₃H⁷, Gln⁴-His⁵, Leu¹⁷-Val¹⁸, His⁵-Leu⁶, Glu¹³-Ala¹⁴, Asn³-Gln⁴).

Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr⁴-Ile⁵ and Phe⁸-His⁹ bonds.     

Initial cleavage site of the oxidized insulin B-chain by a ribosomal proteinase fromStreptococcus lactis,a food microorganism

The unique specificity of a ribosomal proteinase fromStreptococcus lactis, a food microorganism, was investgated with the oxidized insulin B-chain. The initial cleavage site of the enzyme was observed at Leu¹⁵-Tyr¹⁶ bond, and additional site at Arg²²-Gly²³     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.     

Substrate specificity of carboxyl proteinase ofAspergillus sojae

The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu¹⁵-Tyr¹⁶ bond and the Phe²⁴-Phe²⁵ bond. Additional cleavage of the bond Tyr¹⁶-Leu¹⁷ was also noted.     

Specificities of extracellular and ribosomal serine proteinases from Bacillus natto,a food microorganism

The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Ser⁹-His¹⁰ and additional cleavage sites at Gln⁴-His⁵ and His⁵-Leu⁶ in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe⁸-His⁹ and secondary at Tyr⁴-Ile⁵. The extracellular serine proteinase has a K_m of 0.08 mM and k_cat of 3 s⁻¹ for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu¹⁵-Tyr¹⁶ and additional cleavage site at Phe²⁴-Phe²⁵. Hydrolysis of proangiotensin was observed at Tyr⁴-Ile⁵ bond with the ribosomal proteinase.     

Inhibition of Human Pepsin and Gastricsin by α2-Macroglobulin

The inhibitory effects of human α₂-macroglobulin (α₂-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α₂-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α₂-M mainly at the His⁶⁹⁴-Ala⁶⁹⁵ bond and Leu⁶⁹⁷-Val⁶⁹⁸ bond, respectively, in the bait regions sequence of α₂-M. The conformation of α₂-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α₂-M.     

Demonstration of beta-lipotropin activating enzyme in porcine pituitary.

Porcine β-lipotropin was incubated with a crude porcine pituitary homogenate, and the main cleavage products of the hormone were isolated and identified. Our results gave evidence for the enzymic cleavage of the Lys⁴⁶-Met⁴⁷, Arg⁶⁰-Tyr⁶¹, Leu⁷⁷-Phe⁷⁸, and Lys⁷⁹-Asn⁸⁰ bonds of the β-lipotropin structure. The cleavage of the Arg⁶⁰-Tyr⁶¹ peptide bond was accompanied with the concomitant release of opiate activity in the first period of incubation, provided that bacitracin was present in the incubation mixture. The enzyme was differentiated from trypsin or plasmin and appears to be a specific intracellular protease involved in the biosynthesis of pituitary endorphins.     

Purification and properties of a proteinase from Vipera lebetina (snake) venom

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr¹⁶-Leu¹⁷, Phe²⁴-Phe²⁵ and Phe²⁵-Tyr²⁶, and glucagon at the bonds Tyr¹⁰-Ser¹¹, Leu¹⁴-Asp¹⁵ and Leu²⁶-Met²⁷. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.     

Bitter Taste of Synthetic C-Terminal Tetradecapeptide of Bovine β-Casein,H-Pro196-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val209-OH,and Its Related Peptides

The primary structure of bovine β-casein contains the partial sequence of -Pro¹⁹⁶-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val²⁰⁹ in the C-terminal portion. We previously reported that the synthetic C-terminal octapeptide, Arg²⁰²-Val²⁰⁹, is extremely bitter with its threshold value 0.004 mm, 250 times as strong as that of caffeine. To further investigate the bitter taste of the C-terminal portion of β-casein, we synthesized the C-terminal tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, and some of its fragments. A hydrophobic hexapeptide, Pro¹⁹⁶-Val²⁰¹, was twice as bitter as caffeine. The bitter taste of the decapeptide, Pro²⁰⁰-Val²⁰⁹, was the same as that of Arg²⁰²-Val²⁰⁹. Although the tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, was composed of two bitter peptides, Pro¹⁹⁶-Val²⁰¹ and Arg²⁰²-Val²⁰⁹, its bitter taste was weaker than that of Arg²⁰²-Val²⁰⁹ and its threshold value was 0.015 mm. We suggested that the increase of bitterness in peptides through the introduction of hydrophobic amino acids depended on the number of hydrophobic amino acids added. In addition, the synthetic retro analog of Arg²⁰²-Val²⁰⁹ (H-Val-Ile-Ile-Pro-Phe-Pro-Gly-Arg-OH) was not as bitter as Arg²⁰²-Val²⁰⁹. This indicated that the sequence of Arg²⁰²-Val²⁰⁹ is important for extreme bitterness.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.     

Cleavage of Rabbit Myelin Basic Protein by Pepsin

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe⁸⁷-Phe⁸⁸ bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu¹⁵¹-Phe¹⁵² bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe⁴⁴-Phe⁴⁵ and Leu¹⁰⁹-Ser¹¹⁰ bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH₂-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr¹⁴Leu¹⁵ bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.     

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by the angiotensin II analogue Leu⁸ angiotensin II, with a pA₂ value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA₂ value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu⁸ angiotensin II (10^-8 mol·l^-1) and DuP 753 (10^-6 mol·l^-1) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn¹-Val⁵] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu⁸ angiotensin II. The hydrosmotic response to [Asn¹-Val⁵] angiotensin II (10^-7 mol·l^-1) was significantly inhibited by DuP 753 (10^-6 and 5×10^-6 mol·l^-1), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10^-6 mol·l^-1) also inhibited the hydrosmotic response to angiotensin III (10^-7 mol·l^-1). These results suggest that receptors for angiotensin II present in isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.Abbreviations AT ₁ angiotensin receptor subtype 1 - AT ₂ angiotensin receptor subtype 2 - AT II angiotensin II - AT III angiotensin III - CDRC cumulative doseresponse curve(s) - NE norepinephrine - SCC short-circuit current     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Biotechnological production of recombinant analogues of hirudin-1 from <Emphasis Type="Italic">Hirudo medicinalis</Emphasis>

Hirudin-1 is a highly selective inhibitor of thrombin secreted by the salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in the blood coagulation system. Apart from the existing recombinant analogue of hirudin-1 (63-desulfato-hirudin-1) its modified analogues possessing higher activity and stability are of medical value. In this study artificial genes of hirudin-1 and two its analogues ([Leu¹, Thr²]-hirudin-1 and [Leu¹, Thr²]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein DnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogues were developed. The scheme includes the following stages: isolation of the fusion protein, renaturation of the target protein incorporated into the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the peptides obtained was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu¹, Thr²]-63-desulfatohirudin-1 and [Leu¹, Thr²]-63-desulfatohirudin-1/3 allowed us to obtain these peptides with high yields (14, 25 and 24 mg per 1 L of cell culture, respectively) and high activity (13423, 33333 and 19802 ATU/mg, respectively).     

Protective Effect of Some Anions on the Heat-Denaturation of Ovotransferrin

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H₂O₂, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P₁ position, in descending order. The k _cat, K _m and k _cat/K _m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min^?1, 5.1±0.28 μM, and 3.3±0.28 μM^?1 min^?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.     

Occurrence of cyclosporins and cyclosporin-like peptolides in fungi

Apart from 17 previously listed fungal taxa producing cyclosporin A and its natural congeners B to Z, several additional and taxonomically diverse strains producing the single novel component [Thr², Leu⁵, Leu¹⁰]cyclosporin or cyclosporin-like peptolides (eg SDZ 214-103=[Thr², Leu⁵, D-Hiv⁸, Leu¹⁰]cyclosporin) have recently been described. We report here the isolation of two further and novel cyclosporins, [Thr², Leu⁵, Ala¹⁰]cyclosporin (2) and [Thr², Ile⁵] cyclosporin (3), from strains classified asAcremonium luzulae (Fuckel) W Gams andLeptostroma anamorph ofHypoderma eucalyptii Cooke & Harkn, respectively. In both new strains the usual pattern of cyclosporins A to Z is not found. The structure elucidations of2 and3 are based on NMR spectroscopy, and biological data (immunosuppressive activity, cyclophilin-binding affinity and antifungal effects) are presented.     

Analysis of Substrate Specificity and Endopeptidyl Activities of the Cathepsin B-like Proteinase from <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

The cathepsin B-like proteinase from Helicoverpa armigera (HCB) is involved in the degradation of yolk proteins during embryonic development. In order to gain insight into the substrate specificity of this proteinase, various proteins from animals and plants were tested as substrates. The specific cleavage sites of this enzyme on endopeptide bonds were assayed using bovine serum albumin (BSA) as a substrate. Results showed that BSA was degraded into several fragments, which suggests that HCB cleaves BSA at specific endopeptidyl sites. The amino acid sequences of the BSA derived peptides were determined, revealing cleavage of the bonds between residues Arg⁸¹–Glu⁸², Val⁴²³–Glu⁴²⁴ and Gly⁴³⁰–Lys⁴³¹. This suggests that the minimum requirement for a scissile bond to be recognized by HCB is the presence of an ionic amino acid at the P₁^′ position and the P₁ position can vary. These observations suggest that HCB cleaves bonds at the N-terminal side of ionic amino acid residues giving HCB a wide range of substrates, though other factors dictating the substrate specificity of this enzyme remains to be clarified. Our results provide new evidence that HCB functions as an endopeptidase on some proteins.     

Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based polytetra-, polypenta-, and polynonapeptides

Differential scanning calorimetry studies of the effect of NaCl on protein-based polymer self-assembly has been carried out on six elastin-based synthetic sequential polypeptides- i.e., the polypentapeptide (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and its more hydrophobic analogues (L -Leu¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and (L -Val¹-L -Pro²-L -Ala³-L -Val⁴-Gly⁵)_n; the polytetrapeptide (L -Val¹-L -Pro²-Gly³-Gly⁴)_n and its more hydrophobic analogue (L -IIe¹-L -Pro²-Gly³-Gly⁴)_n; and the polynonapeptide (a pentatetra hybrid), (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵-L -Val⁶-L -Pro⁷-Gly⁸-Gly⁹)_n. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order of the polypentapeptide, as the temperature is raised from below to above that of the transition, has been repeatedly observed using different physical characterizations. In the present experiments, it is observed that the transition temperatures of the polypeptides studied are linearly dependent on NaCl concentration. The molar effectiveness of NaCl in shifting the transition temperature ΔT_m/[N], is about 14°C/[N], with the dependence on peptide hydrophobicity being fairly small. Interestingly, however, the δΔQ/ [N] does depend on the hydrophobicity of a polypeptide.     

Studies on Proteinases from Gliocladium roseum

The alkaline proteinases of Gliocladium roseum (Link) Bainier were purified in crystalline forms by procedures of alcoholic precipitation, fuller’s earth- and acrinol-treatment, and isolated in two types. (Proteinases I and II). Both of these proteinases were homogeneous on zone electrophoresis with polyacrylamide gel (Gyanogum 41), and had the optimal pH values of 11 (Proteinase I) and 10 (Proteinase II), and the optimal temperature of 45°C.

The enzymatic reaction of proteinase I was remarkably promoted by Fe⁺⁺ and Co⁺⁺, and that of proteinase II was promoted by Fe⁺⁺, Go⁺⁺ and Ca⁺⁺, and both proteinases were protected from heat-inactivation by Ca⁺⁺ Proteinase II was activated remarkably by Cl^? under the existence of Fe⁺⁺, but proteinase I was unaffected by the anion.

The order of strength of proteolytic power of these proteinases and chymotrypsin on casein was as follows; proteinase I> proteinase II> chymotrypsin.