The purification and polypeptide composition of avian infectious bronchitis virus.

Purification of egg-grown infectious bronchitis virus (IBV) by sucrose density gradient centrifugation alone, or sucrose density gradient centrifugation plus pH 8.0 treatment, concanavalin A precipitation or metrizamide density gradient centrifugation, failed to produce any differences in the virus polypeptide pattern following polyacrylamide gel electrophoresis in the presence of SDS(SDS-PAGE). SDS-PAGE of purified IBV on 7.5% acrylamide gels separated 16 polypeptides which were detectable by staining with Coomassie blue or measurement of radioactivity following electrophoresis of (3H)-leucine labelled IBV. The molecular weights of the polypeptides were within the range 15,000-135,000. The polypeptides of egg and chick kidney (CK) cell-grown IBV were identical in both size and number but quantitative differences were detected. In particular the relative proportion of the major 52,000 molecular weight polypeptide was greatly reduced in IBV grown in CK cells. SDS-PAGE of purified IBV and staining with Schiff's reagent to detect carbohydrate revealed four.bands with molecular weights of 128,000, 86,000, 67,500 and 37,000. The 128,000 band did not correspond to any of the detected polypeptides. Use of 5% acrylamide gels for SDS-PAGE of IBV failed to resolve all the minor polypeptides and only seven bands were detected.     

Purification and characterization of high salt-soluble vicilin from mung bean (Vigna radiata)

We report a method for the purification of vicilin from mung bean (Vigna radiata) mainly on the basis of solubility of mung bean vicilin even in high salt. Mung bean vicilin remains in solution even after 90% relative saturation of ammonium sulphate. The resulting supernatant after dialysis was subjected to gel filtration (Sephadex G-150) to remove other contaminant polypeptides, and finally the protein was purified by DEAE cellulose chromatography. This purified fraction exhibited 3 bands on SDS-PAGE compared with vicilin from other legumes which exhibite more than 3 bands generally. The results raise the possibility that the presence of the two small polypeptides in vicilin preparations is the breakdown product of the major larger one of mol.wt. 52 K and that vicilin may be a tetramer of four subunits of Mr 52000. That the high salt-soluble protein containing 52 K subunit is vicilin has been determined by several criteria.     

Molecular Properties and Extracellular Processing of the Lipase of Staphylococcus warneri M

Staphylococcus warneri M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1-N3) were determined. From the genome library of S. warneri M whole DNA, the gene-directing lipase activity (named gehC(WM)) was cloned and characterized. The gehC(WM )gene encoded a protein (GehC(WM)), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. warneri 863, GehC(WM) differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. warneri strain. The N-terminal sequences of the N1-N3 polypeptides completely coincided with the deduced amino acid sequences in GehC(WM). GehC(WM) was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehC(WM) is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehC(WM) possessed zinc ion. A gehC(WM) knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehC(WM). Zymogram and immunoblot analyses of the gehC(WM )mutant indicated that GehC(WM) was a major extracellular lipase of S. warneri M.     

Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.     

Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle-free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain-coated vesicles by extraction with 0.5 M TRIS-HCl followed by Sepharose CL-4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr approximately 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500-dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+-induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr approximately 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr approximately 50,000 and 16,500 bands. This treatment abolished the in vitro coat-forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr approximately 100,000 band. Disappearance of the Mr approximately 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr approximately 76,000 and 65,000. When the elastase-treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr approximately 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr approximately 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000-dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.     

Proteins Specified by Herpes Simplex Virus XII. The Virion Polypeptides of Type 1 Strains

The polypeptides from purified virions of a herpes simplex 1 (human herpes-virus 1) strain, F1, which had been passaged a limited number of times in cell culture after isolation, formed 33 bands on electrophoretic separation in polyacrylamide gels cross-linked with N, N'-diallyltartardiamide in contrast to a maximum resolution of only 24 to 25 bands in gels cross-linked with N, N'-methylenebisacrylamide. This increase in the number of bands was due chiefly to an improved separation of glycosylated polypeptides from nonglycosylated polypeptides with which they co-electrophoresed on methylenebisacrylamide cross-linked gels. Purified virions of HSV-1 [F1] had a protein/DNA mass ratio of 10.7 +/- 0.96, and based on a DNA molecular mass of 85 x 10(6) to 100 x 10(6) the estimated weight of virion polypeptides ranges from 16.4 to 19.4 x 10(-16) g. The number of molecules of each polypeptide per virion ranged from less than 50 to 1,500. Comparison of the virion polypeptides of two HSV-1 strains with similar isolation and limited passage history with those of four HSV-1 strains with histories of numerous passages outside the human host showed a number of nonrandom variations in virion polypeptides. Thus, although the virion polypeptides of two strains with similar isolation and limited passage history could not be differentiated, strains with extended passage histories differed markedly from each other and from the limited passage strains in the number and electrophoretic mobility of noncapsid polypeptides and notably in those of the envelope.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Hepatitis B surface antigen polypeptides: artifactual bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis caused by aggregation.

Hepatitis B surface antigen, subtype ad, was purified and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two major bands with molecular weights of 23,500 and 27,500 and several weaker bands with higher molecular weights were observed. When the low-molecular-weight bands and the group of high-molecular-weight bands were excised from the gel, eluted, and reelectrophoresed, neither the low-molecular-weight bands nor the high-molecular-weight bands ever appeared alone, but both high- and low-molecular-weight bands always appeared. It was concluded that the apparently high-molecular-weight bands represented aggregates of the two small polypeptides whose monomers formed the major bands. The preparation thus contained only two polypeptides.     

PROTEIN COMPOSITION OF AXONS and MYELIN FROM RAT and HUMAN PERIPHERAL NERVES

Abstract— Proteins of rat and human peripheral nerves were studied in whole nerve homogenates and in purified myelin and axonal preparations of peripheral nerve. Both myelin and axonal fractions were obtained from desheathed and minced nerve segments by flotation and sedimentation, respectively, in 0.85 m -sucrose following hypotonic treatment. The purity of myelin and axonal preparations was confirmed by electron microscopic examination of pelleted material. Nerve proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3 and 7.4. Major protein bands of fresh whole nerve homogenates corresponded to polypeptide bands of either the purified myelin or axon preparations. The most prominent electrophoretic band in peripheral nerve was identified as a myelin glycoprotein with molecular weight of 27,000. The major polypeptides of axon preparations had molecular weights of 200,000, 150,000, 69,000, 55,000 and 27,000. The latter two proteins were believed to represent tubulin and residual major myelin protein, respectively. The three largest axonal polypeptides were believed to be derived from neurofilaments, which represented the predominant organelle of the purified axons. Collagen was also seen in whole nerve homogenates and in purified axons but could be distinguished by its metachromatic staining with Coomassie blue.     

Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components

Bacillus sp. X-b, a biocontrol agent against certain plant pathogenic fungi, secretes a complex of hydrolytic enzymes, composed of chitinase, chitosanase, laminarinase, lipase and protease. Homogenized mycelium of basidiomycete Macrolepiota procera induced activities of these enzymes more effectively than colloidal chitin or partially purified cell walls of another basidiomycete Polyporus squamosus. Subjected to a multi-step purification, the specific activity of chitinase increased 36-fold, chitosanase 69-fold, lipase 44-fold and laminarinase 15-fold. Partially purified chitinase showed two major bands with molecular masses of 46 000 and 35 000 on sodium dodecyl sulfate–polyacrylamide gel electrophoresis while chitosanase and lipase appeared as single bands with molecular masses of 27 000 and 62 000, respectively.     

Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.     

Effect of plasma biotinyl-peptides on biotinidase activity

Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.     

Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum).

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

Some molecular properties of NAD(P)H dehydrogenase from rat liver

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same K(m) value for NADH but different values for V(max.) were obtained for the enzyme purified from Sprague-Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.