Purification of leukocytosis-promoting substance from bovine parotid gland extract

A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 · 10⁴, and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.     

Purification and characterization of phosphofructokinase in bovine parotid gland.

1. Phosphofructokinase (PFK) was purified from bovine parotid gland to 750-fold with the specific activity of 67.5 units/mg protein by Cibacron Blue F3GA affinity chromatography, and TSK DEAE-5PW ion-exchange and TSK G4000SW size exclusion chromatographies on HPLC. 2. On gel-filtration, molecular weight of the native PFK was estimated to 400,000. 3. PFK was a heterotetramer composed of three kinds of subunit with molecular weights of 92,000 (C-type), 88,000 (M-type) and 86,000 (L-type), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometrically, relative amounts of C-, M- and L-type subunit were 1:1:2. 4. Under the physiological conditions of fructose 6-phosphate (Fru-6-P) and ATP concentrations and pH, PFK activity was suppressed and hardly detectable. 5. Fru-6-P relieved PFK from the ATP inhibition. 6. Fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP activated PFK with a reduction of S0.5 for Fru-6-P and subunit cooperativity. Fru-2,6-P2 was more effective than AMP.     

Lymphocyte-increasing action and hypocalcemic action of parotid gland extract.

It has previously been shown in our laboratory that the hypocalcemic substances purified from the thymus have a potent lymphocyte-increasing action in mice. Then, lymphocyte-increasing activity was examined with bovine parotid gland extracts, which showed a hypocalcemic activity as potent as that of the thymus extracts. The lymphocyte-increasing activity was assayed in the littermates of neonatal mice of Swiss-Webster strain; the materials used for this experiment were purified preparations and several fractions obtained from the parotid gland extracts in the course of purification. It was found that the potency of lymphocyte-increasing activity rose approximately in parallel with the rise in hypocalcemic activity with progress in purification. The final product, which was purified from the gland by isoelectric precipitation at pH 5.4, fractional precipitation with ammonium sulfate, chromatography on DEAE-cellulose, gel filtration on Sepharose 6B and preparative disc electrophoresis, gave a single band in polyacrylamide gel electrophoresis. Its intravenous injection in rabbits, in a dose of 10 mug/kg, produced a significant lowering in serum calcium (percent decrease 10.24 +/- 1.06%) compared with control animals given an injection of physiological saline. Intraperitoneal injection of this purified product, in a dose of 0.5 mug/mouse, in the littermates of neonatal mice, also produced a significant increase (ratio of lymphocytes to polymorphs 2.47 +/- 0.07) in lymphocytes compared with control animals injected with physiological saline (L/P ratio 1.57 +/- 0.05). These facts suggest that this purified protein fraction inherently contains both activities, but the possibility cannot be ruled out of slight contamination by a substance having a high activity. On the other hand, a fraction having no activity for lowering serum calcium but which had activity for increasing the lymphocytes was obtained. This is the first paper to report the presence of lymphocyte-increasing substances in the bovine parotid gland and the purification of one of the substances from the gland.     

Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland.

Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

Purification and characterization of an immunomodulatory Peptide from bovine placenta water-soluble extract

An immunomodulatory peptide was isolated from bovine placenta water-soluble extract and purified by consecutive chromatography on DEAE Sepharose CL-6B, Sephadex G-25, and Sephasil C18 column using lymphocyte proliferation assay to identify the active fractions. The immunomodulatory peptide showed a dose-dependent stimulating effect on lymphocyte proliferation. The isoelectric point of the immunodulatory peptide was determined to be 3.82 by capillary isoelectric focusing electrophoresis. The molecular mass of the immunomodulatory peptide was determined to be 2133.52 Da by mass spectrometry. The first 10 amino acid sequence of the immunomodulatory peptide was Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr. This immunomodulatory peptide showed no significant homology with other immunomodulatory peptides. Additionally, it was thermostable, retaining about 60% of immune activity after incubating at 80 degrees C for 30 min.     

Purification and characterization of a novel cytotoxic substance from cell-free extract of Streptococcus pyogenes

A cytotoxic substance designated as streptococcal cytotoxic protein (SCP) was isolated from a cell-free extract of the Su strain of Streptococcus pyogenes possessing cytotoxic and antitumor activity. SCP was purified with a series of column chromatography and preparative PAGE to give a homogeneous single band as revealed by PAGE analysis. The purified SCP has a molecular mass of 165 kDa, composed of four 43 kDa subunits, and its pI is 4.3. SCP was sensitive to proteinases and was labile to heat and at acidic or alkaline pH. SCP showed inhibitory effects on the [3H]thymidine, [3H]uridine and [3H]leucine uptakes and on the growth of cells, and released 51Cr from cells when the protein was added to the cultures of Ehrlich ascites carcinoma (EAC), mouse mammary tumor (MM-2), leukemia (L-1210) and NIH-3T3 mammalian cells in vitro. SCP also showed an antitumor effect on EAC or MM-2 tumor-bearing mice but not on L-1210 tumor-bearing mice in vivo.     

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.     

Studies on salivary gland ribonucleases. II. Purification of ribonucleases from bovine submaxillary gland and the effects of polyamines on their activities.

Four alkaline ribonucleases [EC 3.1.4.22] were purified 2,050- to 3,460-fold from bovine submaxillary gland by repeated CM-Sephadex C-25 chromatography and Sephadex G-50 gel filtration, with a total recovery of about 13%. These were designated as RNase BS1, BS2, BS3, and BS4, based on their order of elution from a CM-Sephadex C-25 column. The molecular weights of these enzymes were estimated by gel filtration to be 19,000, 17,500, 17,000, and 12,000, respectively. These enzymes are very similar to RNase A in that they are inhibited by heparin, show preferential hydrolysis of C5'-O-P linkages adjacent to a cytosine nucleotide rather than a uracil nucleotide, and in their antigenic properties. Spermine was found to stimulate the activities of these enzymes; the degree of stimulation was in the order RNase BS4 greater than BS3 greater than BS2 greater than BS1. The stimulation by spermine is due to the increased cleavage of C5'-O-P linkages adjacent to cytosine nucleotide. The reason for the differences in the degree of spermine stimulation of these enzymes is discussed.     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.