Responsiveness of cardiac Na+ channels to a site-directed antiserum against the cytosolic linker between domains III and IV and their sensitivity to other modifying agents

Elementary Na⁺ currents were recorded in inside-out patches from neonatal rat heart cardiocytes to analyze the influence of a site-directed polyclonal anti-serum against the linker region between the domains III and IV (amino acids 1489–1507 of the cardiac Na⁺ channel protein) on Na⁺ channel gating and to test whether this part of the -subunit may be considered as a target for modifying agents such as the (–)-enantiomer of DPI 201-106.Anti-SLP 1 serum (directed against amino acids 1490–1507) evoked, usually within 10–15 min after cytosolic administration, modified Na⁺ channel activity. Antiserum-modified Na⁺ channels retain a single open state but leave, at –60 mV for example, their conducting configuration consistently with an about threefold lower rate than normal Na⁺ channels. Another outstanding property of noninactivating Na⁺ channels, enhanced burst activity, may be quite individually pronounced, a surprising result which is difficult to interpret in terms of structure function relations. Removal of inactivation led to an increase of reconstructed peak I _Na (indicating a rise in NP _o) and changed I _Na decay to obey second-order kinetics, i.e., open probability declined slowly but progressively during membrane depolarization. The underlying deactivation process is voltage dependent and responds to a positive voltage shift with a deceleration but may operate even at the same membrane potential with different rates. Iodatemodified Na⁺ channels exhibit very similar properties including a conserved conductance. They are likewise controlled by an efficient, voltage-dependent deactivation process. Modification by (–)-DPI 201-106 fundamentally contrasts to the influence of anti-SLP 1 serum and the protein reagent iodate since (–)-DPI-modified Na⁺ channels maintain their open probability for at least 120 msec, i.e., a deactivation process seems lacking. This functional difference suggests that the linker region between the domains III and IV of the -subunit may not be the only target for (–)-DPI 201-106 and related compounds, if at all.This work was supported by a grant of the Deutsche Forschungs-gemeinschaft (Ko 778/2–4), Bonn.     

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

The highly purified DNA Pol- from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231–6237].The catalytic polypeptide, DNA polymerase- of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (M _r = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA- and a complete separation of polymerase complex and primase.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Central effects of TNFα on thermogenesis and fever in the rat

Intracerebroventricular (icv) injection of purified recombinant human tumour necrosis factor (TNF , 4–8g) in conscious rats, produced increases in colonic temperature (1.0°C) and resting oxygen consumption (VO₂, 14%) which were maximal after 80–90 minutes. Pretreatment with propranolol (10mg/kg s.c) significantly inhibited the rise in VO₂, and prevented the increase in body temperature. Icv injection of an antagonist to corticotropin releasing factor (-helical CRF 9-41, 25 g), which prevents the pyrogenic and thermogenic actions of interleukin-1, did not influence the effects of TNF on temperature or VO₂. Injection of a fragment of TNF (113–130 amino acid sequence) did not affect body temperature or VO₂. TNF injection (icv) significantly increased brown adipose tissue (BAT)in vitro mitochondrial GDP binding, and this effect was slightly inhibited, but not prevented, by surgical denervation of the tissue, and was unaffected by pretreatment with -helical CRF 9-41. These data indicate that TNF can stimulate thermogenesis by a direct central action. The effects are largely, but not totally, dependent on the sympathetic nervous system but, unlike the thermogenic actions of interleukin they do not require release of CRF.     

TNF/LTα double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury

Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LT). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LT are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LT knockout (TNF^–/–LT^–/–) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF^–/–LT^–/– mice. Lymphocyte clusters were evident in all TNF^–/–LT^–/– livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF^–/–LT^–/– but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LT signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF^–/–LT^–/– animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Interaction between Duodenase and α1-Proteinase Inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and ₁-proteinase inhibitor (₁-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and ₁-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·10⁵ M^–1·sec^–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and ₁-PI in identical compartments, ₁-PI is suggested to be a duodenase inhibitor in vivo.     

Isolation and characterization of α-amylase derived from starchgrownClostridium acetobutylicum ATCC 824

Summary An extracellular -amylase was purified to homogeneity from the culture supernatant ofClostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified -amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the -1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5–5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2–5 mM) increased -amylase activity by ca. 20%, while the addition of 19 g ml^–1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. TheV _max andK _m of -amylase were 2.17 mg min^–1 and 3.28 mg ml^–1 on amylose, and 1.67 mg min^–1 and 1.73 mg ml^–1 on soluble starch, respectively.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Linkage studies on α-glycerophosphate and isocitrate dehydrogenases in Aedes albopictus (diptera: Culicidae)

Linkage studies were carried out on -glycerophosphate dehydrogenase (-GPDH) and isocitrate dehydrogenase (IDH) in the mosquito Aedes (Stegomyia) albopictus. Only one locus coding for -GPDH was revealed on agar gels by applying adult homogenates. Two loci for IDH were observed using either fourth-instar larvae, pupae, or adults. This study was restricted to the more anodal Idh-2 of the two loci, and -Gpdh. Both -Gpdh and Idh-2 encode dimeric enzymes. Thirteen backcrosses indicated that the -Gpdh and Idh-2 loci are arranged in linkage group 2 in the following order: p (pigmented pupa)—(ca. 2 map units)—Wb (white-body)—(7.5–17.8)—Idh-2—(13.1)—-Gpdh. Females exhibited more recombination than males.This work was supported by a Grant-in-Aid for Co-operative Research from the Ministry of Education, Japan.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA₃). GA₃ greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA₃. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA₃ caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA₃ was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA₃ on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA₃ reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA₃ and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

Cationic oligopeptides modified with lipophilic fragments: Use for DNA delivery to cells

Cationic oligopeptides, including the amphipathic -helical peptides, are applied to the targeted delivery of DNA to eukaryotic cells due to their DNA-compacting properties and the ability to destabilize the cell lipid bilayer in some cases. We synthesized the peptides differing in the number and location of residues of decanoic acid covalently attached to Lys residues in order to combine the DNA-binding and the membrane activities in a single molecule. We chose peptide structures that assisted in the formation of helices. The DNA-binding ability of the peptides and the membrane activity of their complexes with DNA were shown to depend on the structure. The study of erythrocyte hemolysis by complexes with DNA of the pCMV LacZ plasmid and the peculiarities of transfection of these complexes revealed a correlation between the hemolytic activity and the expression level of the lacZ gene in cells.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 22–30.Original Russian Text Copyright © 2005 by Guryanov, Vlasov, Lesina, Kiselev, Baranov, Avdeeva, Vorobev.     

Artificial Proteins with Antiviral Properties of α-Interferons

Consensus 30–36 sequence LKDRHDF of human -interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195–201), already contains the IFN- 130–137 fragment and possesses antiproliferative activity comparable with that of IFN-₂. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-₂. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.     

Conformational stability of α-lactalbumin missing a peptide bond between Asp66 and Pro67

The peptide bond between Asp⁶⁶-Pro67 of -lactalbumin was cleaved with formic acid (cleaved-lactalbumin). Secondary structural changes of the cleaved-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleaved-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism spectrum. The cleaved-lactalbumin contained 29%-helical structure as against 34% for the intact protein. Some helices of the cleaved-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4mM SDS. On the other hand, the helical structures of the cleaved-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no diffrence was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65°C and recovered upon cooling.