An Improved Method for Embedding Brain Tissue in Albumin-Gelatin

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose. and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 μm. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact

Cutting frozen sections of large (greater than 60 cc) blocks of monkey brain using the conventional procedures of infiltration with 30% sucrose as a cryoprotectant before freezing with pulverized dry ice often produces unacceptable levels of freezing artifact (FA) caused by displacement of tissue by ice crystals. Experiments investigating FA utilized perfusion-fixed brains from 46 monkeys and spanned combinations of cryoprotectants (glycerol, sucrose), freezing methods (dry ice or -75 degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's). The effects were evaluated by rating of FA severity in frozen sections of whole monkey brains. Minor FA appears as enlarged capillaries, more serious FA as large vacuoles, and both first appear midway between the periphery and center of the block. Stronger fixatives increased the severity of freezing artifact. The best method for eliminating FA was graded infiltration with up to 20% glycerol and 2% DMSO (in buffer or fixative), followed by rapid freezing in -75 degrees C isopentane. Although using a glycerol-DMSO infiltration before conventional freezing with pulverized dry ice or using conventional sucrose infiltration before freezing in isopentane gave better results than sucrose infiltration and dry-ice freezing, only the combination of glycerol-DMSO infiltration and freezing in isopentane produced consistently excellent results and virtually eliminated freezing artifact. To determine the effect of freezing with dry ice or isopentane on the rate of cooling in large blocks of CNS tissue, thermocouples were embedded in an 80-cc block of albumin-gelatin and frozen with the two methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using isopentane.     

Tissue structure and macromolecular diffusion in umbilical cord. Immobilization of endogenous hyaluronic acid

Diffusion of endogenous hyaluronic acid and 125I-labelled albumin, monitored by desorption from umbilical cord (Wharton's jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role. The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.     

Tissue structure and macromolecular diffusion in umbilical cord immobilization of endogenous hyaluronic acid

Diffusion of endogenous hyaluronic acid and ¹²⁵I-labelled albumin, monitored by desorption from umbilical cord (Wharton's jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover. time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.     

Basic fibroblast growth factor,albumin, and transferrin purified from rat rhodamine fibrosarcoma tissue are all essential for growth of primary tumor cells from the same tissue in serum-free medium

Summary Primary Rhodamine fibrosarcoma (RdF) cells from rats were shown to grow in serum-free medium supplemented with basic fibroblast growth factor (bFGF), albumin, and transferrin, all of which were purified from RdF tissue. Their growth rate with these supplements was similar to that of cells in medium supplemented with calf serum. bFGF purified from RdF tissue (Rd-bFGF), which was previously designated as DNA synthesis factor, stimulated the growth of primary RdF cells maximally at 30 ng/ml in the presence of the other two proteins. Albumin and transferrin were separated from partially purified tumor growth stimulating activity which was previously shown to stimulate growth of primary RdF cells in serum-free medium. The albumin (RdA) and transferrin (RdT) found in the extract of RdF tissue were not due simply to contamination of the tissue with blood, but to their accumulations in the tissue. The growth stimulatory activities of RdA and RdT on primary RdF cells in serum-free medium were maximal at 30 and 10 μg/ml, respectively. These results suggest that Rd-bFGF, RdA, and RdT, all of which accumulate in the tumor tissue, are essential for growth of RdF cells in the tissue. This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture of Japan.     

Growth hormone alters metabolic effects and proteolysis of insulin in adipose tissue during lactation.

To study the regulation of lipogenesis in adipose tissue by insulin and growth hormone during lactation, tissue was biopsied from primiparous bovines at 30 days antepartum and 60 days postpartum. Tissue was cultured for 24 hr or 48 hr in M199 with acetate and glucose, with a change of medium at 24 hr. The three in vitro treatments were: insulin and hydrocortisone at 10 and 50 ng/ml, respectively (IH); IH + 10 ng/ml of growth hormone (G10); and IH + 100 ng/ml of growth hormone (G100). IH allowed lipogenesis rates from 50% to 85% of those in fresh tissue. Addition of 10 ng/ml of growth hormone reduced (P less than 0.05) lipogenesis; at 100 ng/ml, the effect was only slightly greater. The hypothesis that insulin and growth hormone could be degraded by bovine adipose tissue was tested. Adipose tissue cell-free extracts degraded 125I-labeled insulin, but did not degrade labeled growth hormone. The insulin protease activity was further characterized and had a pH optimum of 7.1, a maximum hydrolysis of approximately 70%, and a hydrated molecular mass of approximately 23,000 daltons. Insulin proteolysis was inhibited by specific insulin protease inhibitors and stimulated by disulfide reducing agents. Bovine growth hormone, prolactin, and histone inhibited (P less than 0.05) the proteolysis of insulin, while bovine serum albumin, egg albumin, trypsin inhibitor, and lysozyme did not. Adipose tissue from pregnant and lactating bovines was sensitive to insulin and growth hormone, and growth hormone may modulate activity of an insulin-specific protease.     

Predictive value of microalbuminuria in patients with insulin-dependent diabetes of long duration.

OBJECTIVE--To investigate the predictive value of microalbuminuria (albumin excretion rate 30-300 mg/24 h) as a risk factor for overt diabetic nephropathy in patients with longstanding insulin dependent diabetes. DESIGN--10 year follow up of patients with normoalbuminuria (albumin excretion rate < 30 mg/24 h), microalbuminuria (30-300 mg/24 h), and macroalbuminuria (> 300 mg/24 h) based on two out of three timed overnight urine samples. SETTING--Outpatient clinic of Helsinki University Hospital. SUBJECTS--72 consecutive patients who had had insulin dependent diabetes for over 15 years. MAIN OUTCOME MEASURES--Urinary albumin excretion rate, mortality, and prevalence of diabetic complications after 10 years. RESULTS--56 patients were re-examined at 10 year follow up, 10 had died, five were lost to follow up, and one was excluded because of non-diabetic kidney disease. At initial screening 22 patients had macroalbuminuria, 18 had microalbuminuria, and 26 had normal albumin excretion. Only five (28%, 95% confidence interval 10% to 54%) of the microalbuminuric patients developed macroalbuminuria during the 10 year follow up and none developed end stage renal failure. Two (8%, 1% to 25%) normoalbuminuric patients developed macroalbuminuria and four (15%, 4% to 35%) became microalbuminuric. Seven (32%, 14% to 55%) of the macroalbuminuric patients developed end stage renal failure and six (27%, 11% to 50%) died of cardiovascular complications. CONCLUSION--Microalbuminuria is not a good predictor of progression to overt nephropathy in patients with longstanding insulin dependent diabetes.     

Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats

A major pathway by which cerebrospinal fluid (CSF) is removed from the cranium is transport through the cribriform plate in association with the olfactory nerves. CSF is then absorbed into lymphatics located in the submucosa of the olfactory epithelium (olfactory turbinates). In an attempt to provide a quantitative measure of this transport, 125I-human serum albumin (HSA) was injected into the lateral ventricles of adult Fisher 344 rats. The animals were killed at 10, 20, 30, 40, and 60 min after injection, and tissue samples, including blood (from heart puncture), skeletal muscle, spleen, liver, kidney, and tail were excised for radioactive assessment. The remains were frozen. To sample the olfactory turbinates, angled coronal tissue sections anterior to the cribriform plate were prepared from the frozen heads. The average concentration of 125I-HSA was higher in the middle olfactory turbinates than in any other tissue with peak concentrations achieved 30 min after injection. At this point, the recoveries of injected tracer (percent injected dose/g tissue) were 9.4% middle turbinates, 1.6% blood, 0.04% skeletal muscle, 0.2% spleen, 0.3% liver, 0.3% kidney, and 0.09% tail. The current belief that arachnoid projections are responsible for CSF drainage fails to explain some important issues related to the pathogenesis of CSF disorders. The rapid movement of the CSF tracer into the olfactory turbinates further supports a role for lymphatics in CSF absorption and provides the basis of a method to investigate the novel concept that diseases associated with the CSF system may involve impaired lymphatic CSF transport.     

非洲爪蟾血清白蛋白的分离纯化及胰蛋白酶抑制活性

通过凝胶过滤层析及两步阴离子交换层析,从非洲爪蟾(Xenopus laevis)的血清中获得了其68kDa的血清白蛋白。与大蹼铃蟾血清白蛋白相似,非洲爪蟾血清白蛋白也具有抑制胰蛋白酶的活性,但其抑制活力相对较低,180nmol/L的非洲爪蟾血清白蛋白能抑制84%的胰蛋白酶活性(30nmol/L)。经表面等离子共振法获得了其与胰蛋白酶的结合动力学常数,解离平衡常数KD=1.44×10-6mol/L。经Western blot分析发现,非洲爪蟾的皮肤中也分布有血清白蛋白。推测两栖类动物血清白蛋白具有的胰蛋白酶抑制活性可能是其抵御天敌捕食的一种防御措施。     

Induction of B16 melanoma melanogenesis by a serum-free synthetic medium.

Cultured murine B16 melanoma cells normally grow as spindle-shaped cells firmly attached to tissue culture flasks. Pellets obtained from harvested B16 melanoma cells are white to grey in color. When the same cells were grown in synthetic, serum-free AIM V medium, cellular morphology and pigmentation were radically altered. Within 3 days of subculture in AIM V, cells rounded up and grew in clusters in suspension. Melanin content increased to greater than 30 times and tyrosinase activity was found to be 10-50 times higher in cells grown in AIM V medium compared to those cultured in normal medium. A concomitant increase in the level of immunoreactive tyrosinase was also induced. The individual growth factors and hormones present in AIM V medium were examined to determine which component(s) stimulates melanogenesis. Only those cells grown in the presence of 2.5% human albumin were stimulated to synthesize melanin. These findings suggest that albumin, or a component associated with albumin, has a major effect upon the regulation of melanogenesis in these cells.     

Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia

The beneficial effect of hemodilution on cerebral blood flow (CBF) during focal cerebral ischemia is mitigated by reduced arterial oxygen content (CaO2). In anesthetized cats subjected to permanent middle cerebral artery occlusion, the time course of regional CBF was evaluated after isovolemic exchange transfusion with either albumin or a tetrameric hemoglobin-based oxygen carrier. The transfusion started 30 min after arterial occlusion. We tested the hypothesis that bulk oxygen transport (CBF x CaO2) to ischemic tissue is increased by hemoglobin transfusion at a hematocrit of 18% compared with albumin-transfused cats at a hematocrit of 18% or control cats at a hematocrit of 30% and equivalent arterial pressure. In the nonischemic hemisphere, CBF increased selectively after albumin transfusion, and oxygen transport was similar among groups. In the ischemic cortex, albumin transfusion increased CBF, but oxygen transport was not increased above that of the control group. Hemoglobin transfusion increased both CBF and oxygen transport in the ischemic cortex above values in the control group, but the increase was delayed until 4 h of ischemia. Consequently, acute injury volume measured at 6 h of ischemia was not significantly attenuated. In contrast to the cortex, CBF in the ischemic caudate nucleus was not substantially increased by either albumin or hemoglobin transfusion. Therefore, in a large animal model of permanent focal ischemia in which transfusion starts 30 min after ischemia, tetrameric cross-linked hemoglobin transfusion can augment oxygen transport to the ischemic cortex, but the increase can be delayed and not necessarily provide protection. Moreover, an end-artery region such as the caudate nucleus is less likely to benefit from hemodilution.     

Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

We identified a new property of human albumin. It enhances formation of fine fibril (or leptofibril) structures during fibrin gelation, and by nephelometric and electron microscopic measurements, this property is independent of and synergistic with that of fibrinogen. We examined fibrin aggregation using physiologic temperatures and pH and albumin:fibrin concentration ratios below those at which the known accelerating effect on fibrin aggregation occurs. An albumin concentration dependent decrease in gel turbidity maxima was consistently demonstrable in buffers containing or lacking (2-5 mM) CaCl2. This decrease was shown to be induced by albumin preparations which had been exposed to 2 mM ethylene-diaminetetraacetate disodium salt (EDTA), dialyzed, and tested in EDTA-free buffer. A delay in the onset of aggregation was also shown in calcium-lacking buffers by use of either reaggregating fibrin or fibrinogen aggregated with low (0.01-0.05 unit/mL) thrombin concentrations. Rates of fibrin aggregation as well as those of fibrinopeptide release were not affected by albumin, and the decrease in gel absorbance was demonstrable when solubilized fibrin was reaggregated at all final fibrin concentrations (0.2-4 microM) examined. Computed from wavelength dependence turbidity measurements (1 microM fibrin, I = 0.20), albumin decreased the average mass:length ratio from 8.24 X 10(11) to 4.26 X 10(11) daltons/cm, or from that of an approximately six to a three protofibril-thick strand. It also decreased the mean fibril radius from 48.5 to 36.4 nm but had no effect on fibril density. Electron microscopic measurements of cross-sectional fibril widths, performed on sections of glutaraldehyde-fixed gels, disclosed differences between albumin-containing and control gels which were significant by chi 2 analysis (P greater than 0.001). Fibril groups of 7-20- and 21-40-nm width together comprised 77% of fibrils formed in the presence of albumin (n = 251) compared to 30% of controls (n = 309). Conversely, coarser fibrils of 41-60- and 61-97-nm width together comprised 23% of fibrils formed in the presence of albumin and 70% of controls. This albumin effect was demonstrable by use of different monomeric albumin preparations including defatted, undefatted (unexposed and exposed to 60 degrees C, 10 h), chromatographically [gel exclusion and (diethylaminoethyl)cellulose] pure, S-(carboxymethyl)albumin, and S-(N-ethylsuccinimidyl)albumin. Chromatographically isolated albumin oligomers lacked this property, suggesting that a specific site(s) on albumin was (were) required.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside GM1 with human and fetal calf sera were studied, the following main results being obtained: (a) GM1, upon incubation with both sera gave origin to two GM1-protein complexes, which also occurred after interaction of GM1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore GM1 appeared to specifically bind serum albumin and to form GM1-albumin complexes. (b) GM1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10(-6) M), was time and concentration dependent, and resulted in a relevant degree of GM1 complexation (up to 80% of total GM1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 . 10(-4) M GM1, the ratio between bound and total GM1 increased linearly with increasing GM1 concentration; in the second phase, occurring above 2 . 10(-4) M, the ratio remained practically constant. After these findings it should be expected that GM1, when present in serum containing systems, forms complexes with albumin. This should be approximately considered when studying the effects of exogeneous GM1 in in vivo and in vitro (tissue cultures) systems.     

Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens.

An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlled-atmosphere chamber which was gassed with 95% O2 and 5% CO2. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30 degrees C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[alpha]pyrene, 7,12-dimethylbenz[alpha]anthracene, aflatoxin B1, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro

Albumin was required specifically for penetration of the zona pellucida (less than 10% of eggs fertilized in the absence of albumin), but was not required for capacitation. A similar rate of capacitation was observed in the presence of albumin at concentrations ranging from 30 to 1 mg/ml, while a slightly slower rate was observed in the presence of 0.25 and 0.1 mg albumin/ml. In the absence of albumin, capacitation occurred at a rate which lagged behind that of the albumin-incubated counterparts by about 30 min; once capacitated, the addition of albumin promoted rapid sperm penetration. In albumin-free media (+/- the macromolecule PVA), sperm motility was frequently reduced, with fewer cells exhibiting hyperactivated motility, but improvements were observed after introduction of albumin. Acrosome loss was significantly lower in the absence of albumin, but within 5 min of its addition at concentrations ranging from 30 to 0.1 mg/ml to capacitated sperm suspensions, acrosome loss was stimulated and reached levels similar to those seen in control samples. Therefore, albumin can trigger the acrosome reaction in capacitated spermatozoa. It appears to act by assisting in the removal of a surface-associated inhibitory component, the presence of which stabilizes the sperm membranes and inhibits the acrosome reaction.     

Binding of bismuth to serum proteins: implication for targets of Bi(III) in blood plasma

Bismuth complexes have been widely used in clinical treatment as antiulcer drugs. However, different adverse effects have been observed and the diagnosis is generally confirmed by the detection of bismuth in blood or blood plasma. In this study, binding of bismuth to human serum albumin was studied by fluorescence spectroscopy with the binding constant logK(a) to be 11.2. Competitive binding of bismuth to human albumin and transferrin was carried out at pH 7.4 by FPLC and ICP-MS. It was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin (albumin/transferrin=13:1) at pH 7.4, 10 mM bicarbonate. The distribution of bismuth between the two proteins was almost unchanged when Cys(34) of albumin was blocked. However, all bismuth binds to albumin when iron-saturated transferrin was used. Almost all of the bismuth was distributed over the fractions containing transferrin (70%) and albumin (<30%) in serum. The percentage of bismuth associated with transferrin was further increased by 15% with elevated transferrin in serum. Binding of bismuth to transferrin is much stronger than human albumin. Transferrin is probably the major target of bismuth in blood plasma, and it may play a role in the pharmacology of bismuth.     

Neuropeptide Y is a vasoconstrictor in human nasal mucosa.

Neuropeptide Y (NPY) is a neurotransmitter in sympathetic nerve fibers in human nasal mucosa. Like norepinephrine, NPY acts as a vasoconstrictor. An established method of nasal provocation was used to determine the effects of topically applied NPY on nasal resistance to airflow measured by anterior rhinomanometry, the protein content of nasal secretions, and the protein content of bradykinin-induced secretions. NPY (2.3 nmol) reduced the resistance to inspiratory airflow by 57 +/- 18% (P < 0.001) in 10 normal subjects and by 50 +/- 17% (P < 0.05) in 12 subjects with perennial rhinitis. In nasal provocations, NPY in doses of 0.1-10 nmol had no effect on vascular (albumin), glandular (lysozyme, glycoconjugate), or total proteins present in lavaged nasal secretions. Because the vasoconstrictor properties of NPY may only be apparent in the presence of increased vascular permeability and albumin exudation, bradykinin (BK) nasal provocation was performed. BK (500 nmol) significantly increase total protein (10- to 20-fold), albumin (10- to 30-fold), and glycoconjugate (2- to 5-fold) in lavage fluid. NPY (2.3 nmol) reduced BK-induced total protein by 59 +/- 15% (P < 0.05) and albumin by 63 +/- 17% (P < 0.02) but had no significant effect on glandular secretion. Therefore exogenous administration of NPY to the human nasal mucosa reduced nasal airflow resistance and albumin exudation without affecting submucosal gland secretion. NPY agonists may be useful for the treatment of mucosal diseases characterized by vasodilation, vascular permeability, and plasma exudation.     

Effects of biogenic phosphates on protease-induced protein cleavage and functioning of plasminogen activators

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.     

Effect of epinephrine on acetyl-CoA carboxylase in rat epididymal fat tissue.

If acetyl-CoA carboxylase in epididymal fat tissue is subject to control by convalent modification as in the case of the liver enzyme, catalytically different forms of carboxylase should exist, independent of polymerization. By treating epididymal fat tissue in culture with epinephrine, we have demonstrated catalytically less active forms of acetyl-CoA carboxylase. The catalytically less active forms of the enzyme reacted to antibody with the same efficiency as the active form of carboxylase. However, the less active enzyme formed by epinephrine treatment of tissues has a sedimentation constant of 30 to 35 S, whereas that of the enzyme from control tissue is 45 S. Incubation of the less active forms of the carboxylase with 10 mM citrate and up to 10 mg/ml of bovine serum albumin activated the enzyme without any change in the sedimentation constant. Therefore, the less active forms of the carboxylase formed as a result of epinephrine treatment are not due to the depolymerization of polymeric forms (45 S) to the protomeric forms (17 to 20 S), but to the formation of intermediate species of carboxylase which cannot form polymeric enzyme (45 S) in the presence of high concentrations of citrate.